Virus vectors and methods of making and administering the same

ABSTRACT

The present invention provides genetically-engineered parvovirus capsids and viruses designed to introduce a heterologous gene into a target cell. The parvoviruses of the invention provide a repertoire of vectors with altered antigenic properties, packaging capabilities, and/or cellular tropisms as compared with current AAV vectors.

RELATED APPLICATION INFORMATION

[0001] This application claims the benefit of provisional applicationsSerial No. 60/107,840, filed on Nov. 10, 1998, and Serial No.60/123,651,filed on Mar. 10, 1999, which are incorporated herein by reference intheir entirety.

STATEMENT OF FEDERAL SUPPORT

[0002] This was made, in part, with government support under grantnumbers DK42701 and 5-32938 0-110 from the National Institutes ofHealth. The United States government has certain rights to thisinvention.

FIELD OF THE INVENTION

[0003] The present invention relates to virus vectors, in particular,modified parvovirus vectors and methods of making and administering thesame.

BACKGROUND

[0004] Parvoviruses are small, single-stranded, non-enveloped DNAviruses between twenty to thirty nanometers in diameter. The genomes ofparvoviruses are approximately 5000 nucleotides long, containing twoopen reading frames. The left-hand open reading frame codes for theproteins responsible for replication (Rep), while the right-hand openreading frame encodes the structural proteins of the capsid (Cap). Allparvoviruses have virions with icosahedral symmetry composed of a majorCap protein, usually the smallest of the Cap proteins, and one or twominor Cap proteins. The Cap proteins are generated from a single genethat initiates translation from

[0005] Most parvoviruses have narrow host ranges; the tropism of B19 isfor human erythroid cells (Munshi et al., (1993) J. Virology 67:562),while canine parvovirus has a tropism for lymphocytes in adult dogs(Parrish et al., (1988) Virology 166:293; Chang et al., (1992) J.Virology 66:6858). Adeno-associated virus, on the other hand, canreplicate well in canine, mouse, chicken, bovine, monkey, as well asnumerous human lines, when the appropriate helper virus is present. Inthe absence of helper virus, AAV will infect and establish latency inall of these cell types, suggesting that the AAV receptor is common andconserved among species. Several serotypes of AAV have-been identified,including serotypes 1, 2, 3, 4, 5 and 6.

[0006] Adeno-associated virus (AAV) is a dependent parvovirus twentynanometers in size which requires co-infection with another virus(either adenovirus or certain members of the herpes virus group) toundergo a productive infection in cultured cells. In the absence ofco-infection with helper virus, the AAV virion binds to a cellularreceptor and enters the cell, migrating to the nucleus, and delivers asingle-stranded DNA genome that can establish latency by integrationinto the host chromosome. The interest in AAV as a vector has centeredaround the biology of this virus. In addition to its unique life-cycle,AAV has a broad host range for infectivity (human, mouse, monkey, dog,etc.), is ubiquitous in humans, and is completely nonpathogenic. Thefinite packaging capacity of this virus (4.5 kb) has restricted the useof this vector in the past to small genes or cDNAs. To advance theprospects of AAV gene delivery, vectors sufficient to carry larger genesmust be developed. In addition, virions that specifically andefficiently target defined cell types without transducing others will berequired for clinical application.

[0007] The capsid proteins of AAV2 are Vp1, 2, and 3 with molecularweights of 87, 73, and 62 kDa, respectively. Vp3 represents nearly 80%of the total protein in intact virions, while Vp1 and Vp2 represent 10%each (Muzyczka, (1992) Curr. Topics Microbiol. Immunol 158:97; Rollinget al., (1995) Molec. Biotech. 3:9; Wistuba et al. (1997) J. Virology71:1341). Early studies of AAV2 support that all three capsid subunitsare required to extract single stranded genomes from the pool ofreplicating double stranded DNA. These genomes are then sequestered intopreformed immature particles that maturate to infectious particles.These particles have a density between 1.32 and 1.41 g/mL in cesiumchloride and sediment between 60S and 125S in sucrose (Myers et a/.,(1981) J. Biological Chem. 256:567; Myers et al., (1980) J. Virology35:65).

[0008] Previous mutagenesis studies of AAV2 capsids have shown thatinsertions and deletions in the Vp3 domain completely inhibit theaccumulation of single stranded virions and production of infectiousparticles (Hermonat et al., (1984) J. Virology 51:329; Ruffing et al,(1992) J. Virology 66:6922). Yang et aL, (1998) Human Gene Therapy9:1929, have reported the insertion of a sequence encoding the variableregion of a single chain antibody against human CD34 at the 5′end of theAAV2 Vp1, Vp2 or Vp3 coding regions. These investigators observedextremely low transduction of CD34 expressing KG-1 cells by AAV virionscontaining the Vp2 fusion protein (1.9 transducing units/ml or less,sentence spanning pages 1934-35). KG-1 cells are reportedly notpermissive to infection by a wild-type rAAV vector. These results withthe Vp2 fusion AAV are suspect as transduction of KG-1 cells by thisvirus was essentially insensitive to an anti-AAV capsid antibody (430vs. 310 transducing units/ml; Table 2), whereas transduction of HeLacells was markedly reduced by this antibody (63,2000 vs. <200transducing units/ml; Table 2). No characterization of the putativefusion virions was undertaken to confirm that the particles containedthe Vp2 fusion protein, the antibody was expressed on the capsidsurface, or that the particles bound CD34 proteins. In addition, rAAVparticles could only be produced if all three wild-type capsid subunitswere provided, in addition to the chimeric subunit (Page 1934, Col. 2,lines 5-12). Collectively, these results suggest the chimeric subunitswere not incorporated into viable AAV particles, and the low level ofchimeric protein observed in target cells was, in fact, due to cellularuptake of chimeric capsid protein or protein aggregates by othermechanisms.

[0009] Several studies have demonstrated that parvovirus capsid proteinscan be mutated and virion assembly studied. In one study, the codingregion for 147 amino acids of the hen egg white lysozyme was substitutedfor B19 Vp1 unique coding sequence. This modification resulted inpurified empty particles that retained lysozyme enzymatic activity(Miyamura et al., (1994) Proc. Nat. Acad. Sci. USA 91:8507). Inaddition, expression of peptides (9 and 13 residues) in B19 Vp2 resultedin empty particles that were immunogenic in mice supporting surfacepresentation of the insertions (Brown et aL, (1994) Virology 198:477).In a more recent study, the CD8+CTL epitope (residues 118-132) againstlymphocytic choriomeningitis virus (LCAAV) nucleoprotein was insertedinto the Vp2 capsid protein of porcine parvovirus (ppv) (Sedlik et aL,(1997) Proc. Nat. Acad. Sci. USA 94:7503). This capsid protein, with theepitope cloned at the N-terminus, self-assembled when expressed in abaculovirus system. This chimeric virus-like particle was then used toimmunize mice against a lethal challenge from LCAAV. While these studiesevaluated capsid structure and assembly, they did not address the issueof packaging B19 genomes into the altered capsids.

[0010] Recombinant (r)AAV vectors require only the inverted terminalrepeat sequences in cis of the 4679 bases to generate virus. All otherviral sequences are dispensable and may be supplied in trans (Muzyczka,(1992) Curr. Topics Microbiol. Immunol. 158:97). Attractivecharacteristics of AAV vectors for gene therapy are the stability,genetic simplicity, and ease of genetic manipulation of this virus.While each of these factors remains valid, some obstacles to theapplication of rAAV vectors have recently come to light. These includeinefficiency of vector transduction and packaging constraints. It is notsurprising, given the cryptic nature of this virus, that new insightsinto its biology have surfaced only after extensive research with rAAVvectors, which are more easily assayed compared with wild-type AAV.

[0011] With respect to the efficiency of vector transduction, severalrecent studies have shown great promise in terms of duration oftransgene expression in vivo; however, there has been a shortfall in theefficiency of transduction, which was unexpected based on previousresults in vitro (Flotte et aL., (1993) Proc. Nat. Acad. Sci. USA90:10613). One of the first experiments in rodents to demonstrate theutility of rAAV vectors in vivo was aimed at transduction of braintissue in rat (Kaplitt et a/, (1994) Nature Genet. 7:148). In additionto brain, muscle has been found to be efficiently transduced in vivo byAAV vectors, demonstrating long term gene expression (at least 1.5years), lack of immune response, and no vector toxicity (Xiao et al,(1996) J. Virol 70:8098; Clark et a/, (1996) Hum. Gene Ther. 8:659;Fisher et at, (1997) Nat Med. 3:306; Monahan et al, (1998) Gene Ther.5:40). The primary steps that influence efficient vector delivery arevirus entry and conversion of second strand synthesis (see Ferrari etal., (1996) J. Virology 70:3227-34).

[0012] The overall success of AAV as a general-purpose viral vectordepends on the ability to package larger than full-length AAV genomes (5kb) into rAAV vectors. Studies by Dong et al., (1996) Hum. Gene Ther.7:2101, have determined the packaging limitations using rAAV vectors asbetween 104% and 108%. This packaging restriction precludes the use of anumber of important genes currently being tested for human gene therapy(e.g., the dystrophin gene or current mini-dystrophin constructs).

[0013] Accordingly, there remains a need in the art for improved virusvectors with greater packaging limits and transduction efficiency thanAAV vectors. In addition, there remains a need for virus vectors withaltered tropisms as compared with AAV vectors.

SUMMARY OF THE INVENTION

[0014] The present invention provides parvovirus vectors for introducing(i.e., delivering) and, preferably, expressing a nucleotide sequence ina cell. The invention is based, in part, on the discovery thatparvovirus vectors possessing unique structures and characteristics ascompared with current vectors may be created by substituting orinserting a foreign sequence (i.e., an exogenous amino acid sequence)into a parvovirus capsid. The invention further provides novel vectorsthat are generated by cross-packaging a parvovirus genome (preferably,an AAV genome) within a different parvovirus capsid. The presentinvention provides a repertoire of novel parvovirus vectors that maypossess unique and advantageous antigenic properties, packagingcapabilities, and cellular tropisms as compared with current AAVvectors.

[0015] These and other aspects of the invention are set forth in moredetail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the insertional mutagenesis strategy for the AAV2capsid. A cassette containing the Kan^(r) gene flanked by EcoRV and NaeI sites were cloned into the plasmid pAV2Cap. pAV2Cap, which containsthe open reading frame of AAV2 capsid, was partially digested with HaeIII, Nla IV, and Rsa I separately so that unit length products wereisolated. The 43 positions of restriction sites for these enzymes areshown above the diagram of the capsid open reading frame. The positionof the Kan^(r) insert was mapped by restriction enzyme digestion and insome cases sequenced. Once the position was determined the Kan^(r) genewas removed by EcoRV digestion, and the capsid domain subcloned intopACG. This strategy resulted in inserting a 12 base pair fragment, withhalf Nae I sites flanking a unique Eco RV site, into the respective HaeIII, Nla IV, and Rsa I sites. The twelve base pairs code for four aminoacids one of which is shown above the diagram of pACG2.

[0017]FIG. 2 shows the expression of capsid proteins in cellstransfected with wild-type and insertion mutant helper plasmids ofpACG2. Cell lysates from 293 cells transfected with 1, H2285; 2, H2634;3, H2690; 4, N2944; 5, H2944; 6, H3595; 7, H4047; 8, wild-type wereanalyzed by acrylamide gel electrophoresis and immunoblofting with theBl monoclonal antibody and detected by peroxidase-conjugated secondaryantibody. On the left of the Western blot are the positions of themolecular weight standards and on the right are the positions of themajor capsid protein, VP3 and the minor capsid proteins VP2 and VP1.

[0018]FIG. 3 shows expression of a Lac Z transgene in cells infectedwith insertion mutant or wild-type virus. Panel A. Dot blothybridization to the Lac Z transgene. Cell lysates of adenovirusinfected 293 cells transfected with the insertion mutant or wild-typehelper plasmids and the Lac Z transgene containing vector were subjectedto cesium chloride isopycnic gradient. Fractions from the gradient weretreated with DNase and RNase prior to dot blotting to remove unpackagednucleic acids, fraction numbers are labeled above the dot blot. Fraction1 has a density range of 1.377-1.41, fraction 2 has a density range of1.39-1.435, and fraction 3 has a density range of 1.42-1.45. Theβ-galactosidase gene was used as the control template, to estimateparticle numbers. Estimates of particle number where derived assuming 1μg of 1000 bp DNA has 9.1×10¹¹ molecules. Panel B. Infection of HeLacells with 1.75×10⁸ particles from various insertion mutants andwild-type capsid containing the Lac Z transgene. Cells expressing thetransgene appear blue when stained with X-gal.

[0019]FIG. 4 shows characterization of the insertion mutants usingelectron microscopy. 200 uL samples of each virus from peak fraction ofgradient were dialyzed against 1×PBS+1 mM MgCl₂ and speed-vacdesiccated, then resuspended in 20 uL of distilled H₂O. Samples werenegative stained with 2% phosphotungstic acid. Panel A. rAAV2 withwild-type virion. Infectious insertion viruses H2690 (Panel B), andH2591 (Panel C). Non-infectious viruses H2285 (Panel D), H2634 (Panel E)and, H3595 (Panel F). The black bar is 100 nm; the magnification isequivalent in each panel.

[0020]FIG. 5 presents analysis of virion composition from wild-type andvarious insertion mutant viruses isolated from cell lysates by cesiumchloride gradient centrifugation. Peak fractions of virus weredetermined by dot blot hybridization and dialyzed against 1×PBS+1 mMMgCl₂. Foreach, viral sample between 1.0 ×10⁹ and 2.5×10⁹ particles wereused. Virions from 1. Wild-type rAAV2; 2. H2285; 3. R2349; 4. H2591; 5.H2634; 6. H2690; 7. H3766; and 8. N4160 were analyzed by acrylamide gelelectrophoresis and immunoblofting with the B1 monoclonal antibody anddetected by peroxidase-conjugated secondary antibody. On the left of theWestern blot are the positions of the molecular weight standards and onthe right are the positions of the major capsid protein, VP3 and theminor capsid proteins VP2 and VP1.

[0021]FIG. 6 shows the analysis of wild-type and non-infectiousinsertion mutant virus batch binding to heparin agarose by dot blothybridization. Viruses with wild-type virions and insertion in thecapsids were dialysed against 0.5×PBS and 0.5 mM MgCl₂. One hundredmicroliters of each virus was bound to 100 μl of heparin agarose, atroom temperature for one hour. Samples were washed six times with 500μof wash buffer each, followed by elution with 100 μof 0.5, 1.0 and 1.5MNaCl each, and the supernatant from each wash and elution step wassaved. Twenty microliters of supernatant from each step and 20 μl of theagarose pellet were used for dot blot hybridization. Pairs of washeswere combined and {fraction (1/50)} of the total volume from each pairwas used for dot blot hybridization, while one fifth of the elutionsupernatant and agarose bed volumes were used. The 100% bound wasequivalent to one fifth of the virus added to the heparin agarose.Samples 1. rAAV2 with wild-type virion; 2. H2285; 3. H2416; 4. H2634;and 5. H3761.

[0022]FIG. 7 is schematic representation of the AAV2/4 subunit chimeras.

[0023]FIG. 8 is a diagram of the helper plasmid pAAV2/B19p2Cap. Thecoding region of the B19 major structural protein, Vp2, was seamlesslycloned from pAAV-Vp3 to TAA.

[0024]FIG. 9 provides EM analysis of chimeric virus particles producedwith pAAV/B19Vp2Cap.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides parvovirus vectors for thedelivery of nucleic acids to cells, both in vitro and in vivo.Alternatively, the invention provides novel capsid structures for use,e.g., as vaccines or for delivery of compounds to cells (e.g., asdescribed by U.S. Pat. No. 5,863,541 to Samulski et al., the disclosureof which is incorporated herein by reference in its entirety). Theparvovirus vectors of the present invention utilize the advantageousproperties of AAV vectors, and may mitigate some of the problemsencountered with these vectors. In particular embodiments, theparvovirus vectors may possess different or altered characteristics fromAAV vectors, including but not limited to, antigenic properties,packaging capabilities, and/or cellular tropism.

[0026] The term “parvovirus” as used herein encompasses allparvoviruses, including autonomously-replicating parvoviruses anddependoviruses. The autonomous parvoviruses include members of thegenera Parvovirus, Erythrovirus, Densovirus, Iteravirus, andContravirus. Exemplary autonomous parvoviruses include, but are notlimited to, mouse minute virus, bovine parvovirus, canine parvovirus,chicken parvovirus, feline panleukopenia virus, feline parvovirus, gooseparvovirus, and B19 virus. Other autonomous parvoviruses are known tothose skilled in the art. See, e.g., BERNARD N. FIELDS et al., VIROLOGY,volume 2, chapter 69 (3d ed., Lippincoff-Raven Publishers).

[0027] The genus Dependovirus contains the adeno-associated virusesAAV), including but not limited to, AAV type 1, AAV type 2, AAV type 3,AAV type 4, AAV type 5, AAV type 6, avian AAV, bovine AAV, canine AAV,equine AAV, and ovine AAV. See, e.g., BERNARD N. FIELDS et al.,VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers).

[0028] The parvovirus particles, capsids and genomes of the presentinvention are preferably from AAV.

[0029] The term “tropism” as used herein refers to entry of the virusinto the cell, optionally and preferably, followed by expression ofsequences carried by the viral genome in the cell, e.g., for arecombinant virus, expression of the heterologous nucleotidesequences(s). Those skilled in the art will appreciate thattranscription of a heterologous nucleic acid sequence from the viralgenome may not be initiated in the absence of trans-acting factors,e.g., for an inducible promoter or otherwise regulated nucleic acidsequence. In the case of AAV, gene expression from the viral genome maybe from a stably integrated provirus, from a non-integrated episome, aswell as any other form in which the virus may take within the cell.

[0030] The parvovirus vectors of the present invention are useful forthe delivery of nucleic acids to cells both in vitro and in vivo. Inparticular, the inventive vectors may be advantageously employed todeliver or transfer nucleic acids to animal cells. Nucleic acids ofinterest include nucleic acids encoding peptides and proteins,preferably therapeutic (e.g., for medical or veterinary uses) orimmunogenic (e.g., for vaccines) peptides or proteins.

[0031] A “therapeutic” peptide or protein is a peptide or protein thatmay alleviate or reduce symptoms that result from an absence or defectin a protein in a cell or subject. Alternatively, a “therapeutic”peptide or protein is one that otherwise confers a benefit to a subject,e.g., anti-cancer effects. Therapeutic peptides and proteins include,but are not limited to, CFTR (cystic fibrosis transmembrane regulatorprotein), dystrophin (including the protein product of dystrophinmini-genes, see, e.g, Vincent et al., (1993) Nature Genetics 5:130),utrophin (Tinsley et aL, (1996) Nature 384:349), clotting factors(Factor XIII, Factor IX, Factor X, etc.), erythropoietin, the LDLreceptor, lipoprotein lipase, ornithine transcarbamylase, β-globin,α-globin, spectrin, α-antitrypsin, adenosine deaminase, hypoxanthineguanine phosphoribosyl transferase, β-glucocerebrosidase,sphingomyelinase, lysosomal hexosaminidase, branched-chain keto aciddehydrogenase, hormones, growth factors (e.g., insulin-like growthfactors 1 and 2, platelet derived growth factor, epidermal growthfactor, nerve growth factor, neurotrophic factor −3 and −4,brain-derived neurotrophic factor, glial derived growth factor,transforming growth factor-αand -β, and the like), cytokines (e.g.,α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4,interleukin 12, granulocyte-macrophage colony stimulating factor,lymphotoxin), suicide gene products (e.g., herpes simplex virusthymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450,deoxycytidine kinase, and tumor necrosis factor), proteins conferringresistance to a drug used in cancer therapy, tumor suppressor geneproducts (e.g., p53, Rb, Wt-1, NF1, VHL, APC, and the like), and anyother peptide or protein that has a therapeutic effect in a subject inneed thereof.

[0032] Further exemplary therapeutic peptides or proteins include thosethat may used in the treatment of a disease condition including, but notlimited to, cystic fibrosis (and other diseases of the lung), hemophiliaA, hemophilia B, thalassemia, anemia and other blood disorders, AIDS,Alzheimer's disease, Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, epilepsy, and other neurologicaldisorders, cancer, diabetes mellitus, muscular dystrophies (e.g.,Duchenne, Becker), Gaucher's disease, Hurler's disease, adenosinedeaminase deficiency, glycogen storage diseases and other metabolicdefects, retinal degenerative diseases (and other diseases of the eye),and diseases of solid organs (e.g., brain, liver, kidney, heart).

[0033] The present invention also provides vectors useful as vaccines.The use of parvoviruses as vaccines is known in the art (see, e.g.,Miyamura et al., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Pat. No.5,916,563 to Young et al., 5,905,040 to Mazzara et aL, U.S. Pat. Nos.5,882,652, U.S. Pat. No. 5,863,541 to Samulski et al.; the disclosuresof which are incorporated herein in their entirety by reference). Theantigen may be presented in the parvovirus capsid, as described belowfor chimeric and modified parvovirus vectors. Alternatively, the antigenmay be expressed from a heterologous nucleic acid introduced into arecombinant AAV genome and carried by the inventive parvoviruses. Anyimmunogen of interest may be provided by the parvovirus vector.Immunogens of interest are well-known in the art and include, but arenot limited to, immunogens from human immunodeficiency virus, influenzavirus, gag proteins, tumor antigens, cancer antigens, bacterialantigens, viral antigens, and the like.

[0034] As a further alternative, the heterologous nucleic acid sequencemay encode a reporter peptide or protein (e.g., an enzyme). Reporterproteins are known in the art and include, but are not limited to, GreenFluorescent Protein, β-galactosidase, alkaline phosphatase,chloramphenicol acetyltransferase, and the like.

[0035] Alternatively, in particular embodiments of the invention, thenucleic acid of interest may encode an antisense nucleic acid, aribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs thateffect spliceosome-mediated trans-splicing (Puffaraju et al., (1999)Nature Biotech. 17:246), or other non-translated RNAs, such as “guide”RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat.No. 5,869,248 to Yuan et al.), and the like.

[0036] Except as otherwise indicated, standard methods known to thoseskilled in the art may be used for the construction of rAAV genomes,transcomplementing packaging vectors, transiently and stably transfectedpackaging cells according to the present invention. Such techniques areknown to those skilled in the art. See, e.g., SAMBROOK et aL, MOLECULARCLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989);F. M. AUSUBEL et aL CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(GreenPublishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0037] Hybrid Viruses.

[0038] The hybrid parvovirus vectors of the present invention mayovercome some of the disadvantages of AAV vectors for delivery ofnucleic acids or other molecules to cells.

[0039] A “hybrid” parvovirus, as used herein, is an AAV genomeencapsidated within a different (i.e., another, foreign, exogenous)parvovirus capsid. Alternatively stated, a hybrid parvovirus has aparvovirus genome encapsidated within a different parvovirus capsid. Asused herein, by “different” it is intended that the AAV genome ispackaged within another parvovirus capsid, e.g., the parvovirus capsidis from another AAV serotype or from an autonomous parvovirus.

[0040] Preferably, the parvovirus genome is an AAV genome (preferably arecombinant AAV genome). It is also preferred that the AAV genomecomprises one or more AAV inverted terminal repeat(s) as describedbelow. Typically, as described in more detail below, a recombinant AAV(rAAV) genome will retain only those elements required in cis (e.g., oneor more AAV ITRs), with the rest of the genome (e.g., the rep/cap genes)being provided in trans.

[0041] In particular preferred embodiments the parvovirus capsid is anAAV capsid (i.e., a hybrid AAV vector). According to this embodiment,the AAV capsid packages an AAV genome of a different serotype (andpreferably, of a different serotype from the one or more AAV ITRs). Forexample, a recombinant AAV type 1, 2, 3, 4, 5 or 6 genome may beencapsidated within an AAV type 1, 2, 3, 4, 5 or 6 capsid, provided thatthe AAV capsid and genome (and preferably, the one or more AAV ITRs) areof different serotypes.

[0042] Illustrative hybrid parvoviruses according to the presentinvention are an AAV type 2 genome packaged within an AAV type 1, 3, 4,5 or 6 capsid. In particular preferred embodiments, the hybridparvovirus comprises an AAV type 3, type 4, or type 5 capsid packagingan AAV type 2 genome, more preferably, an AAV type 3 or type 5 capsidpackaging a type 2 genome.

[0043] In other preferred embodiments, an AAV type 1, 3, 4, 5 or 6genome is packaged within a different AAV capsid (e.g., a type 1 genomein a type 2, 3, 4, 5, or 6 capsid, and the like).

[0044] Also preferred are hybrid B19/AAV parvoviruses in which an AAVgenome (e.g., an AAV type 1, 2, 3, 4, 5 or 6 genome) is packaged withina B19 capsid. More preferably, the hybrid parvovirus has a B19 capsidand an AAV type 2 genome.

[0045] Further preferred are hybrid parvoviruses in which a mouse minutevirus, bovine parvovirus, canine parvovirus, chicken parvovirus, felinepanleukopenia virus, feline parvovirus, or goose parvovirus capsidpackages an AAV genome, more preferably an AAV type 2 genome.

[0046] Specific hybrid viruses include those having the capsid sequenceencoded by the AAV2/4 helper plasmid given in Appendix 1 (nucleotides2123 to 4341 of SEQ ID NO:1). This sequence encodes the AAV2 rep genesand AAV4 capsid in a pBluescript backbone. It is also preferred that thehybrid parvovirus having the capsid sequence given by SEQ ID NO:1 is anAAV2 genome. Alternatively, the nucleotide sequence of the AAV4 capsidis substantially homologous to the nucleotide sequence given asnucleotides 2123 to 4341 of SEQ ID NO:1. As a further alternative, thenucleotide sequence of the AAV4 capsid encodes the amino acid sequenceencoded by nucleotides 2123 to 4341 in SEQ ID NO:1. The term“substantially homologous” is as defined hereinbelow.

[0047] One of the limitations of current AAV vectors for gene deliveryis the prevalence of neutralizing antibodies against AAV within thehuman population. For example, it is estimated that 80% of adults areseropositive for AAV type 2. In preferred embodiments, the instantinvention provides hybrid parvovirus vectors that may be advantageouslyemployed to reduce (e.g., diminish, decrease, mitigate, and the like) animmune response in the subject being treated. Thus, for example, a rAAVtype 2 vector genome carrying a heterologous nucleic acid sequence orsequences may be packaged within an AAV type 3 capsid and administeredto a subject who is seropositive for AAV type 2 and cannot neutralizeAAV type 3 virus.

[0048] According to this aspect of the invention, a rAAV genome may bepackaged within any non-homologous parvovirus capsid for delivery to acell, in vitro or in vivo. In preferred embodiments, the AAV genome ispackaged within an array of non-homologous capsids to overcomeneutralizing antibodies and/or or to prevent the development of animmune response. In particular preferred embodiments, the rAAV may bedelivered within a series of hybrid virus particles, so as tocontinually present the immune system with a new virus vector. Thisstrategy will allow for repeated administration without immuneclearance.

[0049] A further limitation encountered with AAV vectors concerns thecellular tropism of this virus. The wild-type tropism of AAV isproblematic both because AAV infects a wide range of cell types andbecause it exhibits no infectivity in other potential target cells ofinterest (e.g, erythroid cells). Autonomous parvoviruses, in contrast,have a narrower cellular tropism. The tropisms of particular autonomousparvoviruses are known to those skilled in the art. Illustrativecellular tropisms of autonomous parvoviruses include: B19 virus(erythroid cells), canine parvovirus (gut epithelium), AAVM(p)(fibroblasts); and goose parvovirus (myocardial lining of the heart).Furthermore, autonomous parvoviruses exhibit a wider range of hostspecies than does AAV, which characteristic may be utilized to developAAV vectors for administration to bovines, canines, felines, geese,ducks, and the like, e.g., for veterinary treatments. Thus,cross-packaging of AAV genomes in autonomous parvovirus capsidsaccording to the present invention may be utilized to produce a virusvector with a different cellular tropism than AAV.

[0050] With respect to AAV/AAV hybrids, all of the AAV serotypes infecta broad host range of cells. However, there are differences in the ratesof vector transduction, suggesting that the different serotypes may usedifferent cellular receptors. In addition, only limited competition isobserved among serotypes in binding experiments, which observationfurther indicates that the different serotypes have evolved to usedistinct receptors (Mizukami et al., (1996) Virology 217:124).Accordingly, hybrid parvoviruses of the present invention that packagean AAV genome in an AAV capsid of a different serotype also provideopportunities for delivering AAV vectors to a wider range of cell typesthan current AAV vectors and/or for directing AAV vectors to specifictarget cells.

[0051] In preferred embodiments, the hybrid parvovirus particle containsa rAAV genome. As used herein, the rAAV genome carries at least oneheterologous nucleic acid sequence to be delivered to a cell. Thoseskilled in the art will appreciate that the rAAV genome can encode morethan one heterologous nucleic acid sequence (e.g., two, three or moreheterologous nucleic acid sequences), generally only limited by thepackaging capacity of the virus capsid. Heterologous nucleic acidsequence(s) of interest for use according to the present invention areas described above.

[0052] As used herein, a recombinant hybrid parvovirus particleencompasses virus particles with hybrid, chimeric, targeted and/ormodified parvovirus capsids as described hereinbelow. Moreover, thoseskilled in the art will understand that the parvovirus capsid mayinclude other modifications or mutations (e.g., deletion, insertion,point and/or missense mutations, and the like). Likewise, the rAAVgenome may include modifications or mutations (e.g., deletion,insertion, point and/or missense mutations, and the like). Those skilledin the art will further appreciate that mutations may incidentally beintroduced into the rAAV genome or parvovirus capsid as a result of thecloning strategy employed.

[0053] The rAAV genome of the hybrid parvovirus preferably encodes atleast one AAV inverted terminal repeat (ITR), preferably two AAV ITRs,and more preferably two homologous AAV ITRs, which flank theheterologous nucleic acid sequence(s) to be delivered to the cell. TheAAV ITR(s) may be from any AAV, with types 1, 2, 3, 4, 5 and 6 beingpreferred, and type 2 being most preferred. The term “inverted terminalrepeat” includes synthetic sequences that function as an AAV invertedterminal repeat, such as the “double-D sequence” as described in U.S.Pat. No. 5,478,745 to Samulski et aL., the disclosure of which isincorporated in its entirety herein by reference. It has beendemonstrated that only a single 165 bp double-D sequence is required incis for site specific integration, replication, and encapsidation ofvector sequences. AAV ITRs according to the present invention need nothave a wild-type ITR sequence (e.g., a wild-type sequence may be alteredby insertion, deletion, truncation or missense mutations), as long asthe ITR functions to mediate virus packaging, replication, integration,and/or provirus rescue, and the like.

[0054] In hybrid parvoviruses according to the present invention, theAAV ITR(s) is different from the parvovirus capsid. Moreover, if thecapsid is an AAV capsid, the capsid and the ITR(s) are of different AAVserotypes. In preferred embodiments, the AAV ITR(s) is from AAV type 2and the parvovirus capsid is an AAV type 3, 4 or 5 capsid, morepreferably an AAV type 3 or 5 capsid. In alternate preferredembodiments, the hybrid parvovirus has a B19 capsid and the AAV ITR(s)is from AAV type 2.

[0055] The rAAV genomes of the invention may additionally containexpression control elements, such as transcription/translation controlsignals, origins of replication, polyadenylation signals, and internalribosome entry sites (IRES), promoters, enhancers, and the like,operably associated with the heterologous nucleic acid sequence(s) to bedelivered to the cell. Those skilled in the art will appreciate that avariety of promoter/enhancer elements may be used depending on the leveland tissue-specific expression desired. The promoter/enhancer may beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer may be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thepromoter/enhancer region is not found in the wild-type host into whichthe promoter/enhancer region is introduced.

[0056] Promoters/enhancers that are native to the target cell or subjectto be treated are most preferred. Also preferred are promoters/enhancersthat are native to the heterologous nucleic acid sequence. Thepromoter/enhancer is chosen so that it will function in the targetcell(s) of interest. Mammalian promoters/enhancers are also preferred.

[0057] Inducible expression control elements are preferred in thoseapplications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery are preferablytissue-specific promoter/enhancer elements, and include muscle specific(including cardiac, skeletal and/or smooth muscle), neural tissuespecific (including brain-specific), liver specific, bone marrowspecific, pancreatic specific, spleen specific, retinal specific, andlung specific promoter/enhancer elements. Other induciblepromoter/enhancer elements include hormone-inducible and metal-inducibleelements. Exemplary inducible promoters/enhancer elements include, butare not limited to, a Tet on/off element, a RU486-inducible promoter, anecdysone-inducible promoter, a rapamycin-inducible promoter, and ametalothionein promoter.

[0058] In embodiments of the invention in which the heterologous nucleicacid sequence(s) will be transcribed and then translated in the targetcells, specific initiation signals are generally required for efficienttranslation of inserted protein coding sequences. These exogenoustranslational control sequences, which may include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

[0059] The AAV genome of the inventive parvovirus vectors may optionallyinclude the genes that encode the AAV Cap and Rep proteins. In preferredembodiments, the genes encoding at least one of the AAV Cap proteins orat least one of the AAV Rep proteins will be deleted from the rAAVgenome. According to this embodiment, the Cap and Rep functions may beprovided in trans, e.g., from a transcomplementing packaging vector orby a stably-transformed packaging cell line. In more preferredembodiments, the genes encoding all of the AAV Cap proteins or all ofthe AAV Rep proteins will be deleted from the rAAV genome. Finally, inthe most preferred embodiments, all of the AAV cap genes and all of theAAV rep genes are deleted from the AAV vector. This configurationmaximizes the size of the heterologous nucleic acid sequence(s) that canbe carried by the AAV genome, simplifies cloning procedures, andminimizes recombination between the rAAV genome and the rep/cappackaging sequences provided in trans.

[0060] In hybrid parvoviruses according to the present invention, theparvovirus cap genes (if present) may encode the Cap proteins from anyparvovirus, preferably an AAV. In contrast, the rep genes (if present)will typically and preferably be AAV rep genes. It is further preferredthat the rep genes and the AAV inverted terminal repeat(s) carried bythe AAV genome are of the same serotype. Moreover, if the cap genes areAAV cap genes, the rep genes will preferably be of a different AAVserotype from the AAV cap genes.

[0061] The rep genes/proteins of different AAV serotypes may beevaluated for those giving the highest titer vector in connection withparticular hybrid parvoviruses without undue experimentation. Inparticular preferred embodiments, the AAV rep genes encode atemperature-sensitive Rep78 and/or Rep68 protein as described by Gavinet al., (1999) J. Virology 73:9433 (the disclosure of which isincorporated herein by reference in its entirety).

[0062] As described above, the Cap proteins of the hybrid parvovirus aredifferent from the AAV genome (i.e., the Cap proteins are either from adifferent AAV serotype or from an autonomous parvovirus). In addition,as described above, the Cap proteins will typically and preferably bedifferent from the rep genes (if present).

[0063] Accordingly, in particular preferred embodiments, the hybridparvovirus has an AAV type 3, 4 or 5 capsid and carries an AAV type 2genome including an AAV type 2 ITR(s). The AAV genome may additionallyinclude the AAV rep genes (preferably type 2) and AAV cap genes(preferably, AAV type 3, 4, or 5, respectively). Typically, however, theAAV genome will be a rAAV genome, and the rep and cap genes will bedeleted therefrom. In an alternate preferred embodiment, the hybridparvovirus has a B19 capsid and carries an AAV genome, more preferablyan AAV type 2 genome, including an AAV ITR(s). The AAV genome mayoptionally encode the AAV Rep proteins (preferably AAV type 2) and B19capsid proteins, but preferably is a rAAV genome lacking thesesequences.

[0064] The present invention also provides nucleotide sequences andvectors (including cloning and packaging vectors) encoding the inventiveAAV genomes and the parvovirus cap gene(s) and the AAV rep gene(s) forproducing the inventive hybrid parvoviruses. As described above, inpreferred embodiments, at least one of the AAV rep genes or one of theAAV cap genes, more preferably all of the AAV rep genes and the AAV capgenes, are deleted from the AAV genome. The Rep and Cap functions may beprovided in trans by packaging vector(s). Multiple packaging vectors(e.g., two, three, etc.) may be employed, but typically and preferablyall of the Rep and Cap functions are provided by a single packagingvector.

[0065] Cloning and packaging vectors may be any vector known in the art.Illustrative vectors include, but are not limited to, plasmids, nakedDNA vectors, bacterial artificial chromosomes (BACs), yeast artificialchromosomes (YACs), and viral vectors. Preferred viral vectors includeAAV, adenovirus, herpesvirus, Epstein-Barr virus (EBV), baculovirus, andretroviral (e.g., lentiviral) vectors, more preferably, adenovirus andherpesvirus vectors.

[0066] The present invention also provides cells containing theinventive vectors. The cell may be any cell known in the art includingbacterial, protozoan, yeast, fungus, plant, and animal (e.g., insect,avian, mammalian) cells.

[0067] Further provided are stably-transformed packaging cells thatexpress the sequences encoding the parvovirus cap gene(s) and/or the AAVrep gene(s) for producing the inventive hybrid parvoviruses. Anysuitable cell known in the art may be employed to express the parvoviruscap and/or rep gene(s). Mammalian cells are preferred (e.g., HeLacells). Also preferred are trans-complementing packaging cell lines thatwill provide functions deleted from a replication-defective helpervirus, e.g., 293 cells or other E1a trans-complementing cells.

[0068] In particular preferred embodiments, at least one of the repgenes or at least one of the cap genes, more preferably all of the capgenes or all of the rep genes are stably integrated into the geneticmaterial of the packaging cell and are expressed therefrom. Typically,and most preferably, all of the parvovirus cap genes and all of the AAVrep genes are stably integrated and expressed by the packaging cell.

[0069] The cap and rep genes and proteins are as described above withrespect to hybrid AAV genomes. Thus, the packaging vector(s) and/orpackaging cell may encode the cap genes from any parvovirus. Preferredare the B19, AAV type 3, AAV type 4 and AAV type 5 cap genes. Likewise,the packaging vector(s) and/or packaging cell may encode the rep genesfrom any parvovirus. Preferably, however, the rep genes will be AAVgenes, more preferably, AAV type 2, AAV type 3, AAV type 4, or AAV type5 rep genes. Most preferably, the rep genes are AAV type 2 rep genes. Inparticular preferred embodiments, the AAV rep sequences encode atemperature-sensitive Rep78 or Rep68 protein as described by Gavin etaL, (1999) J. Virology 73:9433.

[0070] The expression of the cap and rep genes, whether carried by therAAV genome, a packaging vector, or stably integrated into the genome ofa packaging cell may be driven by any promoter or enhancer element knownin the art, as described in more detail above. Preferably, the cap orrep genes (more preferably both) are operably associated with parvoviruspromoters. In the most preferred embodiments, the cap genes and repgenes are operably associated with their authentic promoters (i.e., thenative promoter).

[0071] A previous report indicates that expression of parvovirus capgenes from a B19/AAV type 2 hybrid helper vector cannot be achievedusing authentic promoters. Ponnazhagan et al., (1998)-J. Virology72:5224, attempted to generate a helper vector for producing a B19parvovirus capsid packaging an AAV type 2 genome. These investigatorsreported that virus could not be packaged when the cap genes on thehelper vector were driven by either the authentic AAV p40 or B19 p6promoters. Packaging of virus was only successfully achieved when theCAAV promoter (a strong promoter) was substituted for the authenticpromoters. It appears that the natural regulation of the cap genes wasdisrupted, and cap gene expression was restored only by splitting up therep and cap coding regions and using an exogenous promoter to drive capgene expression.

[0072] Likewise, the cloning strategy proposed by U.S. Pat. No.5,681,731 to Lebkowski et al. for generating hybrid viruses comprisingan autonomous parvovirus capsid encapsidating a rAAV genome (col. 15-16)will fail to result in packaged virus.

[0073] In contrast, the present invention provides hybrid packagingvectors and packaging cells in which parvovirus promoters, preferablythe authentic promoters, may be used to drive expression of theparvovirus cap and rep genes to produce the inventive hybridparvoviruses. Previous efforts to create hybrid parvovirus cap/rep geneconstructs using authentic promoters have not succeeded, at least inpart, because these investigators failed to preserve the integrity ofthe splice sites required for proper processing of the rep genes. Thepresent investigations have utilized a seamless cloning strategy(Stratagene USA) in which the splice sites within the rep genes havebeen preserved. Alternatively, site-directed mutagenesis (or similartechniques) may be used to restore the splice sites to the hybrid virusconstructs.

[0074] The present invention further encompasses methods of producingthe inventive hybrid parvoviruses. Hybrid parvovirus particles accordingto the invention may be produced by introducing an AAV genome to bereplicated and packaged into a permissive or packaging cell, as thoseterms are understood in the art (e.g., a “permissive” cell can beinfected or transduced by the virus; a “packaging” cell is a stablytransformed cell providing helper functions). Preferably, the AAV genomeis a rAAV genome encoding a heterologous nucleic acid sequence(s) thatis flanked by at least one AAV ITR. rAAV genomes, AAV ITRs, andheterologous nucleic acid sequences are all as described in more detailhereinabove. The AAV genome may be provided to the cell by any suitablevector, as described hereinabove.

[0075] Any method of introducing the vector carrying the AAV genome intothe permissive cell may be employed, including but not limited to,electroporation, calcium phosphate precipitation, microinjection,cationic or anionic liposomes, and liposomes in combination with anuclear localization signal. In embodiments wherein the AAV genome isprovided by a virus vector, standard methods for producing viralinfection may be used.

[0076] Any suitable permissive or packaging cell known in the art may beemployed to produce AAV vectors. Mammalian cells are preferred. Alsopreferred are trans-complementing packaging cell lines that providefunctions deleted from a replication-defective helper virus, e.g., 293cells or other E1a trans-complementing cells.

[0077] The AAV genome may contain some or all of the AAV cap and repgenes, as described herein. Preferably, however, some or all of the capand rep functions are provided in trans by introducing a packagingvector(s), as described above, into the cell. Alternatively, the cell isa packaging cell that is stably transformed to express the cap and/orrep genes. Packaging vectors and packaging cells are as describedhereinabove.

[0078] In addition, helper virus functions are provided for the AAVvector to propagate new virus particles. Both adenovirus and herpessimplex virus may serve as helper viruses for AAV. See, e.g., BERNARD N.FIELDS et al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincoft-RavenPublishers). Exemplary helper viruses include, but are not limited to,Herpes simplex (HSV) varicella zoster, cytomegalovirus, and Epstein-Barrvirus. The multiplicity of infection (MOI) and the duration of theinfection will depend on the type of virus used and the packaging cellline employed. Any suitable helper vector may be employed. Preferably,the helper vector(s) is a plasmid, for example, as described by Xiao etal., (1998) J. Virology 72:2224. The vector can be introduced into thepackaging cell by any suitable method known in the art, as describedabove.

[0079] AAV vectors can be produced by any suitable method known in theart. The traditional production of rAAV vectors entails co-transfectionof a rep/cap vector encoding AAV helper and the AAV vector into humancells infected with adenovirus (Samulski et al., (1989) J. Virology63:3822). Under optimized conditions, this procedure can yield up to 10⁹infectious units of rAAV per ml. One drawback of this method, however,is that it results in the co-production of contaminating wild-typeadenovirus in rAAV preparations. Since several adenovirus proteins(e.g., fiber, hexon, etc.) are known to produce a cytotoxic T-lymphocyte(CTL) immune response in humans (Yang and Wilson, (1995) J. Immunol.155:2564; Yang et al., (1995) J. Virology 69:2004; Yang et al., (1994)Proc. Nat. Acad. Sci. USA 91:4407), this represents a significantdrawback when using these rAAV preparations (Monahan et al, (1998) GeneTherapy 5:40).

[0080] AAV vector stocks free of contaminating helper virus may beobtained by any method known in the art. For example, AAV and helpervirus may be readily differentiated based on size. AAV may also beseparated away from helper virus based on affinity for a heparinsubstrate (Zolotukhin et al (1999) Gene Therapy 6:973). Preferably,deleted replication-defective helper viruses are used so that anycontaminating helper virus is not replication competent. As a furtheralternative, an adenovirus helper lacking late gene expression may beemployed, as only adenovirus early gene expression is required tomediate packaging of AAV virus. Adenovirus mutants defective for lategene expression are known in the art (e.g., ts100 K and ts149 adenovirusmutants).

[0081] A preferred method for providing helper functions throughinfectious adenovirus employs a non-infectious adenovirus miniplasmidthat carries all of the helper genes required for efficient AAVproduction (Ferrari et aL., (1997) Nature Med. 3:1295; Xiao et al.,(1998) J. Virology 72:2224). The rAAV titers obtained with adenovirusminiplasmids are forty-fold higher than those obtained with conventionalmethods of wild-type adenovirus infection (Xiao et al., (1998) J.Virology 72:2224). This approach obviates the need to performco-transfections with adenovirus (Holscher et aL, (1994), J. Virology68:7169; Clark et al., (1995) Hum. Gene Ther. 6:1329; Trempe and Yang,(1993), in, Fifth Parvovirus Workshop, Crystal River, Fla.).

[0082] Other methods of producing rAAV stocks have been described,including but not limited to, methods that split the rep and cap genesonto separate expression cassettes to prevent the generation ofreplication-competent AAV (see, e.g., Allen et al., (1997) J. Virol.71:6816), methods employing packaging cell lines (see, e.g., Gao et al.,(1998) Human Gene Therapy 9:2353; Inoue et al., (1998) J. Virol.72:7024), and other helper virus free systems (see, e.g., U.S. Pat. No.5,945,335 to Colosi).

[0083] Accordingly, the AAV genome to be packaged, parvovirus cap genes,AAV rep genes, and helper functions are provided to a cell (e.g., apermissive or packaging cell) to produce AAV particles carrying the AAVgenome. The combined expression of the rep and cap genes encoded by theAAV genome and/or the packaging vector(s) and/or the stably transformedpackaging cell results in the production of a hybrid parvovirus in whicha parvovirus capsid encapsidates an AAV genome. The hybrid parvovirusparticles are allowed to assemble within the cell, and are thenrecovered by any method known by those of skill in the art.

[0084] The reagents and methods disclosed herein may be employed toproduce high-titer stocks of the inventive parvovirus vectors.Preferably, the parvovirus stock has a titer of at least about 10⁵transducing units (tu)/ml, more preferably at least about 10⁶ tu/ml,more preferably at least about 10⁷ tu/ml, yet more preferably at leastabout 10⁸ tu/ml, yet more preferably at least about 10⁹ tu/ml, still yetmore preferably at least about 10¹⁰ tu/ml, still more preferably atleast about 10¹¹ tu/ml, or more.

[0085] Alternatively stated, the parvovirus stock preferably has a titerof at least about 1 tu/cell, more preferably at least about 5 tu/cell,still more preferably at least about 20 tu/cell, yet more preferably atleast about 50 tu/cell, still more preferably at least about 100tu/cell, more preferably still at least about 250 tu/cell, mostpreferably at least about 500 tu/cell, or even more.

[0086] It is also preferred that the parvovirus is produced atessentially wild-type titers.

[0087] Those skilled in the art will appreciate that the instantinvention also encompasses hybrid parvovirus vectors that containchimeric capsids and/or capsids that have been modified by insertion ofan amino acid sequence(s) into the capsid to confer altered tropisms orother characteristics, each as discussed in more detail below. The viruscapsids may also include other modifications, e.g., deletion, insertion,point and/or missense mutations, and the like.

[0088] Those skilled in the art will further appreciate that mutationsmay incidentally be introduced into the cap and/or rep genes as a resultof the particular cloning strategy employed. For example, theconstruction of sequences encoding hybrid parvovirus genomes asdescribed above may result in chimeric rep genes (and proteins) becauseof the overlap of the rep and cap sequences (e.g., the cap genes and3′end of the rep genes may be AAV type 3, and the remainder of the repgenes may be AAV type 2). As described above, chimeric AAV rep genes inwhich the 3′region is derived from an autonomous parvovirus willgenerally not function as the splicing signals are not conserved amongAAV and the autonomous parvoviruses, unless site-directed mutagenesis,or a similar technique, is employed to restore the splice sites to thehybrid virus constructs.

[0089] II. Chimeric Viruses

[0090] The present invention further provides the discovery thatchimeric parvoviruses may be constructed that possess unique capsidstructures and characteristics. The strategy described above focused onaltering AAV virus structure and function by cross-packaging AAV genomeswithin different parvovirus capsids. Further diversity in virusparticles may be achieved by substituting a portion of the parvoviruscapsid with a portion of a capsid(s) from a different (i.e., another orforeign) parvovirus(es). Alternatively, a portion of a differentparvovirus capsid(s) may be inserted (i.e., rather than substituted)into the parvovirus capsid to create a chimeric parvovirus capsid. Alsodisclosed are vectors, packaging cells, and methods for constructingchimeric parvovirus particles. The chimeric parvoviruses disclosedherein may possess new antigenic properties, packaging capabilities,and/or cellular tropisms. The chimeric capsids and virus particles ofthe invention are also useful for raising chimera-specific antibodiesagainst the novel capsid structures.

[0091] Parvoviruses, AAV, and rAAV genomes are as described above withrespect to hybrid parvoviruses.

[0092] As used herein, a “chimeric” parvovirus is a parvovirus in whicha foreign (i.e., exogenous) capsid region(s) from a differentparvovirus(s) is inserted or substituted into the parvovirus capsid.Preferably the foreign capsid region is substituted for one of thenative parvovirus capsid regions. In particular embodiments, the foreigncapsid region is swapped for the homologous capsid region within theparvovirus capsid. It is also preferred that the parvovirus capsid is anAAV capsid. According to this embodiment, the AAV capsid may be of anyAAV serotype (e.g., type 1, type 2, type 3, type 4, type 5, type 6,etc., as described above). More preferably, the AAV capsid is an AAVtype 2, type 3, type 4, or type 5 capsid, most preferably an AAV type 2capsid.

[0093] Those skilled in the art will appreciate that the chimericparvovirus may additionally be a hybrid parvovirus (as described above)or may be a targeted, or otherwise modified, parvovirus (as describedbelow). Those skilled in the art will further appreciate that due to theoverlap in the sequences encoding the parvovirus capsid proteins, asingle insertion or substitution may affect more than one capsidsubunit.

[0094] The foreign parvovirus capsid region may be from any parvovirus(i.e., an autonomous parvovirus or dependovirus) as described above.Preferably, the foreign capsid region is from the human B19 parvovirusor from AAV type 3, type 4, or type 5.

[0095] The foreign parvovirus capsid region may constitute all orsubstantially all of a capsid subunit(s) (i.e., domain, for example theVp1, Vp2 and Vp3 subunits of AAV or the Vp1 and Vp2 subunits of B 19virus) or a portion of a capsid subunit. Conversely, more than oneforeign capsid subunit may be inserted or substituted into theparvovirus capsid. Likewise, a portion of a parvovirus capsid subunit orone or more parvovirus capsid subunits may be replaced with one or moreforeign capsid subunits, or a portion thereof. Furthermore, the chimericparvovirus capsid may contain insertions and/or substitutions at morethan one site within the capsid. According to this embodiment, themultiple insertions/substitutions may be derived from more than oneparvovirus (e.g., two, three, four, five or more). Generally, it ispreferred that at least one subunit from the parvovirus capsid isretained in the chimeric capsid, although this is not required.

[0096] In particular embodiments of the invention, the foreignparvovirus capsid region that is inserted or substituted into the nativeparvovirus capsid is at least about 2, 5, 10, 12, 15, 20, 30, 50, or 100amino acids in length.

[0097] The inventive chimeric parvoviruses may contain any parvovirusgenome, preferably an AAV genome, more preferably a recombinant AAVgenome. Embodiments wherein the AAV genome is packaged within a chimericAAV capsid of the same serotype is also preferred. AAV type 2 genomesare most preferred regardless of the composition of the chimericparvovirus capsid.

[0098] In preferred embodiments of the invention, the chimericparvovirus comprises an AAV capsid, more preferably an AAV type 2capsid, in which a capsid region from a B19 parvovirus has beensubstituted for one of the AAV capsid domains. In other preferredembodiments, the chimeric parvovirus comprises an AAV capsid (morepreferably, an AAV type 2 capsid) in which the Vp3 subunit of the AAVcapsid has been replaced by the B19 Vp2 subunit.

[0099] In alternative preferred embodiments, the chimeric parvoviruscomprises an AAV capsid (preferably type 2) in which the Vp1 and Vp2subunits are replaced by the Vp1 subunit of a B19 parvovirus.

[0100] In other preferred embodiments, the chimeric parvovirus comprisesan AAV type 2 capsid in which the type 2 Vpl subunit has been replacedby the Vp1 subunit from an AAV type 1, 3, 4, 5, or 6 capsid, preferablya type 3, 4, or 5 capsid. Alternatively, the chimeric parvovirus has anAAV type 2 capsid in which the type 2 Vp2 subunit has been replaced bythe Vp2 subunit from an AAV type 1, 3, 4, 5, or 6 capsid, preferably atype 3, 4, or 5 capsid. Likewise, chimeric parvoviruses in which the Vp3subunit from an AAV type 1, 3, 4, 5 or 6 (more preferably, type 3, 4 or5) is substituted for the Vp3 subunit of an AAV type 2 capsid arepreferred. As a further alternative, chimeric parvoviruses in which twoof the AAV type 2 subunits are replaced by the subunits from an AAV of adifferent serotype (e.g., AAV type 1, 3, 4, 5 or 6) are preferred. Inexemplary chimeric parvoviruses according to this embodiment, the Vp1and Vp2, or Vp1 and Vp3, or Vp2 and Vp3 subunits of an AAV type 2 capsidare replaced by the corresponding subunits of an AAV of a differentserotype (e.g., AAV type 1, 3, 4, 5 or 6). Likewise, in other preferredembodiments, the chimeric parvovirus has an AAV type 1, 3, 4, 5 or 6capsid (preferably the type 2, 3 or 5 capsid) in which one or twosubunits have been replaced with those from an AAV of a differentserotype, as described above for AAV type 2.

[0101] In still other preferred embodiments, the minor subunit of oneparvovirus may be substituted with any minor subunit of anotherparvovirus (e.g., Vp2 of AAV type 2 may be replaced with Vp1 from AAVtype 3; Vp1 of B19 may substitute for Vp1 and/or VP2 of AAV). Likewise,the major capsid subunit of one parvovirus may be replaced with themajor capsid subunit of another parvovirus.

[0102] The nucleotide sequence of specific chimeric capsids includethose encoded by the helper plasmid given in Appendix 2 (nucleotides2133 to 4315 of SEQ ID NO:2). This sequence contains the AAV2 rep codingsequences, most of the AAV2 Vp1 and Vp3 coding sequences, and the entireAAV4 Vp2 coding sequences and some of the AAV4 Vp1 and Vp3 codingsequences in a pBluescript backbone. Preferably, the chimericparvoviruses having the capsid encoded by the helper given in SEQ IDNO:2 carry an AAV2 genome.

[0103] Alternatively, the nucleotide sequence of the chimeric capsid issubstantially homologous to the capsid coding sequence given asnucleotides 2133 to 4315 of SEQ ID NO:2. As a further alternative, thenucleotide sequence of the chimeric capsid encodes the same amino acidsequence as nucleotides 2133 to 4315 of SEQ ID NO:2. The term“substantially homologous” is as defined hereinbelow.

[0104] The present invention also provides the discovery that chimericparvoviruses may generate unique capsid structures that do not resemblethe constituent parvovirus capsids. For example, the presentinvestigations have discovered that B19/AAV type 2 chimeras, in whichthe Vp3 subunit of AAV type 2 has been replaced by the Vp2 subunit of ahuman B19 virus, results in the expected 23-28 nm particle (typical forwt AAV) and a novel 33-38 nm particle. The larger particles were presentat the same density as the 23-28 nm particles in a cesium isopycnicgradient.

[0105] While not wishing to be held to any particular theory of theinvention, these results suggest that this particle is formed bychanging the triangulation number from T=1 to T=3, to yield a largerparticle containing 180 copies of the major capsid component instead of60. This novel,particle may package larger than wild-type genomes due toits increased size. In particular preferred embodiments, the B19/AAVtype 2 chimeric parvovirus capsid (B19 Vp2 swapped for AAV2 Vp3) has thesequence given as SEQ ID NO. 3 (Appendix 3).

[0106] The present invention further provides B19/AAV chimeric capsidsand parvoviruses having larger than wild-type capsid structures (e.g.,larger than about 28 nm, 30 nm, 32 nm, 34 nm, 36 nm, 38 nm, 40 nm ormore in diameter). Alternatively stated, the present invention providesB19/AAV chimeric capsids and parvoviruses with capsid structurescontaining more than the wild-type number of capsid subunits (e.g.,greater than about 60 capsid subunits, greater than about 90 capsidsubunits, greater than about 120 capsid subunits, greater than about 180capsid subunits). As a further alternative statement, the presentinvention provides B19/AAV capsids and parvoviruses that efficientlypackage greater than wild-type genomes (e.g., greater than about 4.8 kb,5.0 kb, 5.2 kb, 5.4 kb, 5.6 kb, 5.8 kb, 6.0 kb, 6.2 kb, 6.4 kb, 6.6 kb,6.8 kb or more). Preferably, the larger genomes are efficiently packagedto produce viral stocks having the titers described hereinabove.

[0107] It is also preferred that the B19/AAV chimeras have alteredantigenic properties. In particular, it is preferred that the B19/AAVchimeras may be administered to a subject that has antibodies againstthe serotype of the AAV without immune clearance, i.e., the chimera isnot recognized by the AAV serotype-specific antibodies.

[0108] In other preferred embodiment of the invention, the nucleotidesequence of the B19/AAV chimeric capsid is substantially homologous tothe sequence given as SEQ ID NO:3 and encodes a chimeric parvoviruscapsid. This definition is intended to include AAV of other serotypesand non-human B19 viruses. As used herein, sequences that are“substantially homologous” are at least 75%, and more preferably are80%, 85%, 90%, 95%, or even 99% homologous or more.

[0109] High stringency hybridization conditions that permit homologousnucleotide sequences to hybridize are well known in the art. Forexample, hybridization of homologous nucleotide sequences to hybridizeto the sequence given SEQ ID NO:3 may be carried out in 25% formamide,5×SSC, 5×Denhardt's solution, with 100 μg/ml of single stranded DNA and5% dextran sulfate at 42° C., with wash conditions of 25% formamide,5×SSC, 0.1% SDS at 42° C. for 15 minutes, to allow hybridization ofsequences of about 60% homology. More stringent conditions arerepresented by a wash stringency of 0.3M NaCl, 0.03 M sodium citrate,0.1% SDS at 600 or even 70° C. using a standard in situ hybridizationassay. (See SAMBROOK ETAL., MOLECULAR CLONING, A LABORATORY MANUAL (2ded. 1989)).

[0110] In other preferred embodiments, the chimeric B19/AAV capsid hasthe amino acid sequence encoded by the sequence given in SEQ ID NO:3(Appendix 4; SEQ ID NO:4).

[0111] In other particular preferred embodiments, a non-conservedregion(s) of a parvovirus capsid is inserted or substituted, preferablysubstituted, into another parvovirus capsid. Preferably a non-conservedregion(s) is substituted for the same (i.e., homologous) region from adifferent parvovirus. Parvovirus specific (including AAV serotypespecific) characteristics are likely associated with such non-conservedregions. It is also likely that non-conserved regions can, best toleratealterations. In particular embodiments, the looped-out regions of theparvovirus major capsid subunits are swapped between two parvoviruses,more preferably an AAV and a parvovirus, still more preferably betweentwo AAV of different serotypes.

[0112] With particular respect to AAV type 2, although the crystalstructure of this virus has not been solved, structural correlationshave been made based on sequence information. The structuralcorrelations suggest that the Vp3 subunit of AAV type 2 has eightP-barrel motifs, and that these motifs are separated by looped outregions (Chapman et al., Virology 194:419). Recently, the sequence ofAAV type 3 has been determined by Muramatsu et aL., (1996) Virology221:208. The amino acid homology between Vp3 of AAV type 2 and AAV type3 is 89%, with the region defined as loop 3/4 having 70% homology (Id.).Additionally, AAV type 3 does not bind to the same receptor as AAV type2 (Mizukami et aL, Virology 217:124). The divergent amino acid sequencesin loops 3 and 4 may explain the differences in cellular receptors usedby AAV type 2 and AAV type 3, and the resulting disparities in cellulartropism. Accordingly, in preferred embodiments of the instant invention,chimeric AAV particles are constructed in which loop 3/4, or a portionthereof, of AAV type 2 is swapped for the AAV type 3 loop 3/4, or viceversa.

[0113] In other embodiments, the chimeric parvovirus comprises an AAVtype 2 capsid in which loop 1, 2, 3, and/or 4 of the Vp3 subunit havebeen replaced by the corresponding loop region(s) of an AAV of adifferent serotype (e.g., type 1, 3, 4, 5 or 6). In illustrativeembodiments, the loop 2-4 region of the AAV type 2 Vp3 subunit isreplaced by the loop 2-4 region of a type 3 or type 4 virus.

[0114] Likewise, in other preferred embodiments, the chimeric parvoviruscomprises an AAV type 1, 3, 4, 5 or 6 capsid in which the loop 1, 2, 3and/or 4 region of the Vp3 subunit is replaced by the correspondingregion of a different AAV serotype. Exemplary embodiments include, butare not limited to, a chimeric parvovirus comprising an AAV type 3 ortype 4 capsid in which the loop 2-4 region of the Vp3 subunit isreplaced by the AAV type 2 loop 2-4 region.

[0115] The present invention further provides chimeric parvovirusescomprising an AAV capsid in which a loop region(s) in the major Vp3subunit is replaced by a loop region (s) (preferably, a correspondingloop region(s)) from the major subunit of an autonomous parvovirus. Inparticular, the loop region 1, 2, 3 and/or 4 from an AAV type 1, 2, 3,4, 5, or 6 Vp3 subunit is replaced with a loop region from the majorsubunit of an autonomous parvovirus.

[0116] The nucleotide sequence of specific chimeric capsids includethose having the capsid sequence encoded by the helper plasmid given inAppendix 5 (nucleotides 2133 to 4342 of SEQ ID NO:5). This sequencecontains the AAV2 rep coding sequences, most of the AAV2 capsid codingsequences, with the exception that loops 2-4 from the AAV2 Vp3 subunitwere replaced with the corresponding region from AAV3, in a pBluescriptbackbone.

[0117] Alternatively, the nucleotide sequence of the chimeric capsid issubstantially homologous to the sequence given as nucleotides 2133 to4342 of SEQ ID NO:5. As a further alternative, the nucleotide sequenceof the chimeric capsid has the same amino acid sequence as the capsidencoded by nucleotides 2133 to 4342 of SEQ ID NO:5. The term“substantially homologous” is as defined hereinabove.

[0118] Chimeric parvoviruses may be constructed as taught herein or byother standard methods known in the art. Likewise, those skilled in theart may evaluate the chimeric parvoviruses thus generated for assembly,packaging, cellular tropism, and the like, as described herein or byother standard methods known in the art, without undue experimentation.

[0119] Another aspect of the present invention is a chimeric parvoviruscapsid protein (preferably an AAV Vp-1, Vp2 or Vp3 capsid protein) withat least one capsid region from another parvovirus(es) inserted orsubstituted therein (preferably, substituted). The introduction of theforeign capsid protein into a parvovirus capsid provides alteredcharacteristics (e.g., immunogenic, tropism, etc.) to a virus capsid orparticle (preferably a parvovirus capsid or particle) incorporating thechimeric parvovirus capsid protein. Alternatively, the chimericparvovirus capsid protein may facilitate detection or purification of avirus capsid or particle (preferably parvovirus capsid or particle)incorporating the chimeric parvovirus capsid protein. In particularpreferred embodiments, the antigenic properties of an AAV capsid orparticle of a particular serotype may be altered (e.g., changed ormodified) or diminished (e.g., reduced or mitigated) by incorporation ofthe chimeric parvovirus capsid region for the native capsid region.According to this embodiment, chimeric capsid proteins may be used toobviate or reduce immune clearance in subjects that have immunityagainst the serotype of the AAV capsid or particle (e.g., to permitmultiple virus administrations). Changes or reductions in antigenicproperties may be assessed, e.g., in comparison to an AAV capsid orparticle that is identical except for the presence of the chimericparvovirus capsid protein.

[0120] The present invention also encompasses empty chimeric parvoviruscapsid structures. Empty capsids may be used for presentation ordelivery of peptides or proteins (e.g., antigens to produce an immuneresponse), nucleic acids, or other compounds (see, e.g., Miyamura etal., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Pat. No. 5,916,563 toYoung et a/L, 5,905,040 to Mazzara et al., U.S. Pat. Nos. 5,882,652,5,863,541 to Samulski et aL; the disclosures of which are incorporatedherein in their entirety by reference). Empty capsids may be produced byany method known in the art. (see, e.g., id.).

[0121] The chimeric parvoviruses and capsids of the invention furtherfind use in raising antibodies against the novel capsid structures.Antibodies may be produced by methods that are known to those skilled inthe art.

[0122] The present invention also provides cloning vectors,transcomplementing packaging vectors, packaging cells, and methods forproducing the inventive chimeric parvovirus particles disclosed herein.In general, vectors, packaging cells, and methods for producing chimericparvoviruses are as described above with respect to hybrid parvoviruses.In addition, at least one of the cap genes (encoded by the rAAV genome,a packaging vector(s), or the packaging cell) has inserted therein atleast one nucleic acid sequence encoding a foreign amino acid sequencefrom a non-homolgous parvovirus (as described above).

[0123] III. Targeted Parvoviruses

[0124] A further aspect of the present invention are parvovirus vectorscomprising a parvovirus capsid and a recombinant AAV genome, wherein anexogenous targeting sequence has been inserted or substituted into theparvovirus capsid. The parvovirus vector is preferably targeted (i.e.,directed to a particular cell type or types) by the substitution orinsertion of the exogenous targeting sequence into the parvoviruscapsid. Alternatively stated, the exogenous targeting sequencepreferably confers an altered tropism upon the parvovirus. As yet afurther alternative statement, the targeting sequence increases theefficiency of delivery of the targeted vector to a cell.

[0125] As, is described in more detail below, the exogenous targetingsequence may be a virus capsid sequence (e.g., an autonomous parvoviruscapsid sequence, AAV capsid sequence, or any other viral capsidsequence) that directs infection of the parvovirus to a particular celltype(s). As an alternative, the exogenous amino acid sequence may encodeany peptide or protein that directs entry of the parvovirus vectors intoa cell(s). In preferred embodiments, the parvovirus capsid is an AAVcapsid, more preferably, an AAV type 2 capsid.

[0126] An “altered” tropism, as used herein, includes reductions orenhancements in infectivity with respect to a particular cell type(s) ascompared with the native parvovirus lacking the targeting sequence(s).An “altered” tropism also encompasses the creation of a new tropism(i.e., the parvovirus would not infect a particular cell type(s) to asignificant or, alternatively, a detectable extent in the absence of theexogenous amino acid sequence). Alternatively, an “altered tropism” mayrefer to a more directed targeting of the parvovirus vector to aparticular cell type(s) as compared with the native parvovirus, but thetarget cells may typically be infected by the native parvovirus as well(e.g., a narrowed tropism). As a further alternative, an “altered”tropism refers to a more efficient delivery of a targeted parvovirus ascompared with the native parvovirus (e.g., a reduced Multiplicity ofInfection, “MOI”).

[0127] The term “reduction in infectivity”, as used herein, is intendedto encompass both an abolishment of the wild-type tropism as well as adiminishment in the wild-type tropism or infectivity toward a particularcell type(s). The diminished infectivity may be a 25%, 50%, 75%, 90%,95%, 99%, or more decrease in infectivity with respect to the wild-typelevel of infectivity. By “enhancement in infectivity”, it is meant thatthe infectivity with respect to a particular cell type(s) is increasedabove that observed with the wild-type parvovirus, e.g., by at least25%, 50%, 75%, 100%, 150%, 200%, 300%, or 500%, or more.

[0128] The exogenous targeting sequence(s) may replace or substitutepart or all of a capsid subunit, alternatively, more than one capsidsubunit. As a further alternative, more than one exogenous targetingsequence (e.g., two, three, four, five or more sequences) may beintroduced into the parvovirus capsid. In alternative embodiments,insertions and substitutions within the minor capsid subunits (e.g., Vp1and Vp2 of AAV) are preferred. For AAV capsids, insertions orsubstitutions in Vp2 or Vp3 are also preferred.

[0129] Those skilled in the art will appreciate that due to the overlapin the sequences encoding the parvovirus capsid proteins, a singleinsertion or substitution may affect more than one capsid subunit.

[0130] As described above, in particular embodiments, the presentinvention provides chimeric parvovirus particles with unique structuresand properties. The substitution and/or insertion of one or moreparvovirus capsid region(s) for another to create a chimeric parvoviruscapsid may result in the loss of the wild-type parvovirus tropism and/orthe development of a new tropism associated with the exogenous capsidregion(s). Accordingly, targeted parvoviruses may also be chimericparvoviruses as is described in more detail hereinabove. In particular,targeted chimeric parvoviruses are provided in which a capsid subunit(s)or a loop region(s) from the major capsid subunit has been replaced witha capsid subunit(s) or loop region from another parvovirus.

[0131] Accordingly, in particular embodiments of the instant invention,chimeric parvovirus particles are constructed in which the capsiddomains that encode the wild-type parvovirus tropism are swapped withcapsid regions or subunits from a different parvovirus sequence, therebydiminishing or even completely abolishing the wild-type tropism. Theseinfection-negative parvoviruses find use as templates for creatingparvoviruses with targeted tropisms. In this manner, a parvovirus with anew or directed tropism, but lacking the wild-type tropism, may begenerated.

[0132] In another preferred embodiment, a parvovirus capsid region thatdirects the native or wild-type tropism is swapped with a capsid domainthat directs the tropism of another parvovirus, thereby diminishing orablating the native tropism and concurrently conferring a new tropism tothe chimeric parvovirus. In other embodiments, the foreign capsid regionis substituted or inserted into the parvovirus capsid without reducingor extinguishing the wild-type tropism. As a further alternative, morethan one foreign parvovirus capsid region (e.g., two, three, four, five,or more) is swapped into the parvovirus capsid. For example, a firstforeign capsid region may replace the native capsid region directing thewild-type tropism. Additional foreign capsid regions provide thechimeric capsid with a new tropism(s).

[0133] Heparan sulfate (HS) has recently been identified as a primaryreceptor for AAV (Summerford and Samulski, (1998) J. Virology 72:1438).Thus, the capsid structure may be modified to facilitate or enhancebinding of AAV to the cellular receptor or to inhibit or prevent bindingthereto. To illustrate, the tropism of the AAV may be altered byswapping out the HS binding domain for the AAV capsid, for example, withsequences from other parvoviruses that do contain this HS binding domainor any other sequences.

[0134] Several consensus sequences have been identified among ligandsthat bind to HS receptors. In general, HS appears to bind to sequencesincluding clusters of basic amino acids. Illustrative consensussequences include but are not limited to BBXB, BBBXXB, and RX₇FRXKKXXXK,where B is a basic amino acid, and X is any amino acid. Three sequencescontaining clusters of basic amino acids are present in the first 170amino acid residues of the VP1 capsid protein of AAV type 2 as follows:RX₅KKR at amino acids 116 to 124, KX₄KKR at amino acids 137 to 144, andKX₆RKR at amino acids 161 to 170 (AAV type 2 sequence and numbering asdescribed by Srivastava et al., (1983) J. Virology 45:555, as modifiedby Ruffing et aL, (1994) J. Gen. Virology 75:3385, Muzyczka, (1992)Curr. Topics Microbiol. Immunol. 158:97, and Cassinofti et al., (1988)Virology 167:176). In addition, the consensus sequence (RX₇FRPKRLNFK) isfound in the VP1 capsid subunit of AAV type 2 at amino acids 299 to 315.

[0135] It appears that AAV serotypes 4 and 5 do not bind to cellular HSreceptors, or do so with a low efficiency. Accordingly, in particularembodiments, the HS binding domain of AAV serotypes 1, 2, 3, or 6 may bereplaced with the corresponding region of AAV serotype 4 or 5 to reduceor abolish HS binding. Likewise, HS binding may be conferred upon AAVserotype 4 or 5 by inserting or substituting in the HS binding domainfrom AAV 1, 2, 3 or 6.

[0136] The HS consensus sequences are marked by an abundance of basicamino acids. There is a high density of positively charged amino acidswithin the first 170 residues of the AAV type 2 Vp1 Cap protein,including three strings of basic amino acids, which may be involved inan ionic interaction with the cell surface. Accordingly, in oneparticular embodiment of the invention, the affinity of an AAV capsidfor HS receptors is reduced or eliminated by creating a targetedparvovirus in which some or all of the basic sequences are substitutedby other sequences, e.g., from another parvovirus that does not containthe HS binding domain.

[0137] Alternatively, the respiratory syncytial virus heparin bindingdomain may be inserted or substituted into a virus that does nottypically bind HS receptors (e.g., AAV 4, AAV5, B19) to confer heparinbinding to the resulting mutant.

[0138] B19 infects primary erythroid progenitor cells using globoside asits receptor(Brown et al, (1993) Science 262:114). The structure of B19has been determined to 8 A resolution (Agbandje-McKenna et al., (1994)Virology 203:106). The region of the B19 capsid that binds to globosidehas been mapped between amino acids 399-406 (Chapman et al., (1993)Virology 194:419), a looped out region between β-barrel structures E andF (Chipman et al, (1996) Proc. Nat Acad. Sci. USA 93:7502). Accordingly,the globoside receptor binding domain of the B19 capsid may beinserted/substituted into other parvovirus capsids (preferably an AAVcapsid, more preferably, the AAV type 2 capsid) to target the resultingchimeric parvovirus to erythroid cells.

[0139] In more preferred embodiments, the exogenous targeting sequencemay be any amino acid sequence encoding a peptide or protein, which isinserted or substituted into the parvovirus capsid to alter the tropismof the parvovirus. The native parvovirus tropism may be reduced orabolished by insertion or substitution of the amino acid sequence.Alternatively, the insertion or substitution of the exogenous amino acidsequence may target the parvovirus to a particular cell type(s). In yetfurther preferred embodiments, an exogenous targeting sequence issubstituted or inserted into the parvovirus capsid to concurrentlyablate the wild type tropism and to introduce a new tropism. Forexample, a targeting peptide may be inserted directly into a targetingregion of the AAV capsid to simultaneously disrupt the native tropism(e.g., by interfering with binding to-cellular heparan sulfatereceptors) and to direct the targeted AAV vector to particular cells.

[0140] Those skilled in the art will appreciate that the native tropismof a parvovirus may be reduced or abolished without substituting orinserting an exogenous targeting sequence directly into those regions ofthe parvovirus capsid responsible for the receptor binding. Mutants thathave lost the wild-type tropism are useful as templates for the creationof parvoviruses with novel tropisms as taught herein. It is preferredthat substitutions or insertions that result in the loss of wild-typetropism act at the level of receptor binding and/or entry into the cell.In other words, it is preferred that the altered parvovirus is otherwisecapable of infecting a cell if entry into the cell is provided by othermeans, e.g., by a bispecific antibody, by targeting peptide or proteinas disclosed herein, or by any other means known in the art.

[0141] The exogenous targeting sequence may be any amino acid sequenceencoding a protein or peptide that alters the tropism of the parvovirus.In particular embodiments, the targeting peptide or protein may benaturally occurring or, alternately, completely or partially synthetic.Exemplary peptides and proteins include ligands and other peptides thatbind to cell surface receptors and glycoproteins, such as-RGD peptidesequences, bradykinin, hormones, peptide growth factors (e.g., epidermalgrowth factor, nerve growth factor, fibroblast growth factor,platelet-derived growth factor, insulin-like growth factors I and II,etc.), cytokines, melanocyte stimulating hormone (e.g., α, β or γ),neuropeptides and endorphins, and the like, and fragments thereof thatretain the ability to target cells to their cognate receptors. Otherillustrative peptides and proteins include substance P, keratinocytegrowth factor, neuropeptide Y, gastrin releasing peptide, interleukin 2,hen egg white lysozyme, erythropoietin, gonadoliberin, corticostatin,β-endorphin, leu-enkephalin rimorphin, α-neo-enkephalin, angiotensin,pneumadin, vasoactive intestinal peptide, neurotensin, motilin, andfragments thereof as described above. As a further alternative, thetargeting peptide or protein may be an antibody or Fab fragment thatrecognizes, e.g., a cell-surface epitope, such as an anti-receptorantibody. As yet a further alternative, the binding domain from a toxin(e.g., tetanus toxin or snake toxins, such as α-bungarotoxin, and thelike) can be used to target the inventive parvovirus vectors toparticular target cells of interest. In a yet further preferredembodiment the parvovirus vectors may be delivered to a cell using a“nonclassical” importexport signal peptide (e.g., fibroblast growthfactor-1 and -2, interleukin 1, HIV-1 Tat protein, herpes virus VP22protein, and the like) as described by Cleves, (1997) Current Biology7:R318. Also encompassed are peptide motifs that direct uptake byspecific cells, e.g., a FVFLP peptide motif triggers uptake by livercells. Phage display techniques, as well as other techniques known inthe art, may be used to identify peptides that recognize, preferablyspecifically, any cell type of interest.

[0142] The term “antibody” as used herein refers to all types ofimmunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodiesmay be monoclonal or polyclonal and may be of any species of origin,including (for example) mouse, rat, rabbit, horse, or human, or may bechimeric antibodies. Also encompassed by the term “antibody” arebispecific or “bridging” antibodies as known by those skilled in theart.

[0143] Antibody fragments within the scope of the present inventioninclude, for example, Fab, F(ab)2, and Fc fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments may be produced by known techniques.

[0144] The targeting sequence may alternatively encode any peptide orprotein that targets the parvovirus particle to a cell surface bindingsite, including receptors (e.g., protein, carbohydrate, glycoprotein orproteoglycan), as well as any oppositely charged molecule (as comparedwith the targeting sequence or the parvovirus capsid), or other moleculewith which the targeting sequence or targeted parvovirus interact tobind to the cell, and thereby promote cell entry. Examples of cellsurface binding sites include, but are not limited to, heparan sulfate,chondroitin sulfate, and other glycosaminoglycans, sialic acid moietiesfound on mucins, glycoproteins, and gangliosides, MHCl glycoproteins,carbohydrate components found on membrane glycoproteins, including,mannose, N-acetyl-galactosamine, N-acetyl -glucosamine, fucose,galactose, and the like.

[0145] As yet a further alternative, the targeting sequence may be apeptide or protein that may be used for chemical coupling (e.g., throughamino acid side groups of arginine or lysine residues) to anothermolecule that directs entry of the parvovirus into a cell.

[0146] In other embodiments, the exogenous targeting sequence issubstituted or inserted into the capsid to disrupt binding to cellularreceptors (e.g., HS receptor) and/or entry into the cell. For example,the exogenous amino acid sequence may be substituted or inserted intothe region(s) of the AAV capsid that binds to cellular receptors and/orotherwise mediates entry of the virus into the cell. Preferably, theexogenous targeting sequence is inserted into the capsid region(s) thatinteract with cellular HS receptors (as described above). Oneillustrative insertion mutant that forms intact AAV virions yet fails tobind heparin agarose or infect Hela cells is an AAV type 2 mutantgenerated by insertion of an amino acid sequence at bp 3761 of the AAVtype 2 genome (within the Vp3 cap gene region).

[0147] In a further alternative embodiment, the exogenous amino acidsequence inserted into the parvovirus capsid may be one that facilitatespurification of the parvovirus. According to this aspect of theinvention, it is not necessary that the exogenous amino acid sequencealso alters the tropism of the modified parvovirus. For example, theexogenous amino acid sequence may include a poly-histidine sequence thatis useful for purifying the parvovirus over a nickel column, as is knownto those skilled in the art. Alternatively, the region of the AAV capsidthat interacts with heparin and/or heparan sulfate may be substituted orinserted into a parvovirus capsid so that the parvovirus may be purifiedby binding to heparin, e.g., as described by Zolotukhin et aL, (1999)Gene Therapy 6:973, the disclosure of which is incorporated herein inits entirety by reference.

[0148] In other embodiments, the amino acid sequence encodes anantigenic peptide or protein that may be employed to purify the AAV bystandard immunopurification techniques. Alternatively, the amino acidsequence may encode a receptor ligand or any other peptide or proteinthat may be used to purify the modified parvovirus by affinitypurification or any other techniques known in the art (e.g.,purification techniques based on differential size, density, charge, orisoelectric point, ion-exchange chromatography, or peptidechromatography).

[0149] In yet other embodiments of the invention, an amino acid sequencemay be inserted or substituted into a parvovirus particle to facilitatedetection thereof (e.g., with a antibody or any other detection reagent,as is known in the art). For example, the “flag” epitope may be insertedinto the parvovirus capsid and detected using commercially-availableantibodies (Eastman-Kodak, Rochester, N.Y.). Detectable viruses finduse, e.g., for tracing the presence and/or persistence of virus in acell, tissue or subject.

[0150] In still a further embodiment, an exogenous amino acid sequenceencoding any antigenic protein may be expressed in the modified capsid(e.g., for use in a vaccine).

[0151] As described below and in Table 1, the present investigationshave used insertional mutagenesis of the capsid coding sequence of AAVserotype 2 in order to determine positions within the capsid thattolerate peptide insertions. Viable mutants were identified withinsertions throughout each of the capsid subunits. These insertionmutants find use for any purpose in which it is desirable to insert apeptide or protein sequence into an AAV capsid, e.g., for purifyingand/or detecting virus, or for inserting an antigenic peptide or proteininto the capsid. The nucleotide positions indicated in Table 1 (seeExamples) are the positions at which the restriction sites were made,e.g., the new sequences start at the next nucleotide. For example, foran insertion mutant indicated in Table 1 as having an insertion atnucleotide 2285, the new insertion sequence would begin at nucleotide2286.

[0152] It is preferred to insert the exogenous amino acid sequencewithin the parvovirus minor Cap subunits, e.g., within the AAV Vp1 andVp2 subunits. Alternately, insertions in Vp2 or Vp3 are preferred. Alsopreferred are insertion mutations at nucleotide 2285, 2356, 2364, 2416,2591, 2634, 2690, 2747, 2944, 3317, 3391, 3561, 3595, 3761, 4046, 4047,and/or 4160 within the AAV type 2 cap genes, preferably, to generate anAAV type 2 vector with an altered tropism as described herein (AAV type2 numbering used herein is as described by Srivastava et al., (1983) J.Virology 45:555, as modified by Ruffing et al., (1994) J. Gen. Virology75:3385, Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97, andCassinofti et al., (1988) Virology 167:176).

[0153] Insertions at these nucleotide positions for AAV2 will give riseto amino acid insertions following amino acid 28 (nu 2285), 51 (nu2356), 54 (nu 2364), 71 (nu 2416), 130 (nu 2591), 144 (nu 2634), 163 (nu2690), 182 (nu 2747), 247 (nu 2944), 372 (nu 3317), 396 (nu 3391), 452(nu 3561), 464 (nu 3595), 520 (nu 3761), 521 (nu 3766), 615 (nu 4046 and4047), and 653 (nu 4160) within the AAV2 capsid coding region (using thestarting methionine residue for Vp1 as amino acid 1), or thecorresponding regions of AAV of other serotypes as known by thoseskilled in the art. Those skilled in the art will appreciate that due tothe overlap in the AAV capsid coding regions, these insertions may giverise to insertions within more than one of the capsid proteins (Table2). TABLE 2 Insertion Positions in AAV2 CaDsid^(1, 2) Insertion site Vp1Vp2 Vp3 (nucleotide) (amino acid) (amino acid) (amino acid) 2285  28 — —2356  51 — — 2364  54 — — 2416  71 — — 2591 130 — — 2634 144  7 — 2690163  26 — 2747 182  45 — 2944 247 110  45 3317 372 235 170 3391 396 259194 3561 452 315 250 3595 464 327 262 3753 517 380 315 3761 520 383 3183766 521 384 319 3789 529 392 327 3858 552 415 350 3960 586 449 384 3961586 449 384 3987 595 458 393 4046 615 478 413 4047 615 478 413 4160 653516 451

[0154] Alternatively, the exogenous amino acid sequence is inserted atthe homologous sites to those described above in AAV capsids of otherserotypes as known by those skilled in the art (see, e.g., Chiorini etal., (1999) J. Virology 73:1309). The amino acid positions within theAAV capsid appear to be highly, or even completely, conserved among AAVserotypes. Accordingly, in particular embodiments, the exogenous aminoacid sequence is substituted at the amino acid positions indicated inTable 2 (new sequence starting at the next amino acid) in AAV other thanserotype 2 (e.g., serotype 1, 3, 4, 5 or 6).

[0155] As further alternatives, an exogenous amino acid sequence may beinserted into the AAV capsid at the positions described above tofacilitate purification and/or detection of the modified parvovirus orfor the purposes of antigen presentation, as described above.

[0156] One particular AAV type 2 mutant is produced by inserting anamino acid sequence at nucleotide position 2634 of the genome (withinthe Vp2 cap gene region; AAV2 numbering as described above). This mutantforms AAV type 2 virions with normal morphology by electron microscopyanalysis in the absence of detectable expression of the Vp1 and Vp2subunits. Moreover, this mutant protects the viral genome and retainsbinding to a heparin-agarose matrix, although it does not demonstrateinfectivity in HeLa cells. This mutant is useful for administration tosubjects to avoid an immune response against the Vp1 and Vp2 subunits.It further finds use for insertion of large peptides or proteins intothe AAV capsid structure. As one illustrative example, the adenovirusknob protein is inserted into this mutant to target the virus to theCoxsackie adenovirus receptor (CAR).

[0157] Another particular AAV type 2 insertion mutant is produced byinsertion of an exogenous amino acid sequence at bp 3761 of the genome(within the Vp3 capsid coding region). This mutant protects the viralgenome and forms morphologically normal capsid structures, but does notbind heparin-agarose and fails to infect HeLa cells. This mutant isparticularly useful as a reagent for creating AAV vectors lacking thenative tropism. For example, a new targeting region may be introducedinto this mutant at bp 3761 or at another site. As shown in Table 1, thepresent investigations have discovered a variety of positions within theAAV capsid that tolerate insertion of exogenous peptides and retaininfectivity (e.g., at bp 2356, 2591, 2690, 2944, 3595, and/or 4160 ofthe AAV type 2 genome).

[0158] In other preferred embodiments, AAV vectors with multipleinsertions and/or substitutions are created to provide AAV vectorsexhibiting a desired pattern of infectivity, e.g., a non-infectiousinsertion/substitution mutation and an infectious mutation (e.g., asshown in Table 1) may be combined in a single AAV vector. As oneillustrative example, a peptide insertion may be made at bp 3761 of theAAV type 2 genome (within the Vp3 subunit) to create a non-infectiousheparin binding negative mutant. A second peptide insertion may be madeat bp 2356 (alternatively, bp 2591, 2690, 2944, 3595 or 4160) to targetthe vector. The inserted peptide may be one that directs the AAV type 2vector to target cells of interests. In particular embodiments,bradykinin may be inserted at any of the foregoing sites to target thevector to lung epithelial cells (e.g., for the treatment of cysticfibrosis or other lung disorders) or the adenovirus knob protein may beinserted at the foregoing sites to target the vector to cells expressingCAR receptors. Alternatively, this vector may be employed for antigenpresentation to produce an immune response.

[0159] In other embodiments, the substitution or insertion (preferablyinsertion) is made at nucleotides 3789 or 3961 of the AAV2 genome (e.g.,new sequence would start at nu 3790 and 3962, respectively), or thecorresponding site of other AAV serotypes as known by those skilled inthe art. These positions correspond to insertions following amino acid529 and 586, respectively, of the AAV2 capsid (Met #1 of Vp1 as aminoacid 1; Table 2). In particular embodiments, there will be missensemutation at nucleotides 3790-3792 (Glu→lie) or at nucleotides 3960-3961(Gly→Val), respectively, due to the creation of a restriction site aspart of the cloning strategy. In preferred embodiments of the invention,a targeting insertion at nu 3789 or 3961 is combined with the 3761mutation, which results in loss of heparin binding, to create a targetedcapsid or parvovirus.

[0160] In other preferred embodiments an insertion or substitution(preferably, insertion) is made in the AAV2 capsid at nucleotides 3753,3858, 3960, or 3987 (new sequence beginning at the next nucleotide), orthe corresponding sites in AAV of other serotypes. These sitescorrespond to insertions or substitutions following amino acids 517,552, 586, or 595, respectively, of the AAV2 capsid (Met #1 of Vp1 asamino acid 1; Table 2), or the corresponding sites in AAV capsids ofother serotypes as known by those skilled in the art.

[0161] In other preferred embodiments, the insertion or substitution ismade following amino acid 517, 529, 552, 586 or 595 of AAV capsids ofother serotypes, e.g. (1, 2, 3, 5 or 6).

[0162] There is no particular lower or upper limit to the length of theamino acid sequence that may be inserted or substituted into the viruscapsid, as long as the targeted or modified parvovirus capsid retainsthe desired properties (e.g., assembly, packaging, infectivity). Theexogenous amino acid sequence may be as short as 100, 50, 20,16, 12, 8,4 or 2 amino acids in length. Similarly, the exogenous amino acidsequence to be inserted/substituted into the parvovirus capsid may be aslong as 2, 5, 10,12, 15, 20, 50,100, 200, 300 or more amino acids. Inparticular embodiments, the exogenous amino acid sequence encodes anentire protein. Preferably, the exogenous amino acid sequence that isinserted/substituted into the parvovirus capsid is expressed on theoutside surface of the modified parvovirus capsid.

[0163] The present invention further provides targeted parvovirus capsidproteins, whereby a targeting sequence(s) is inserted or substitutedinto a parvovirus capsid protein, as described above. The targetedparvovirus capsid protein confers an altered tropism upon a virus vectoror virus capsid (preferably, a parvovirus vector or capsid)incorporating the targeted parvovirus capsid protein therein as comparedwith the tropism of the native virus vector or virus capsid in theabsence of the targeted parvovirus capsid protein. Likewise, modifiedcapsid proteins (modifications as described above for parvoviruses) areanother aspect of the invention. The modified capsid protein may beincorporated into a parvovirus capsid or particle, e.g., to facilitatepurification and/or detection thereof or for the purposes of antigenpresentation.

[0164] Further provided are targeted and/or modified parvovirus capsidsas described in more detail above in connection with chimeric parvoviruscapsids. In particular embodiments, the present invention providestargeted parvovirus “capsid vehicles”, as has been described for AAVcapsids, e.g., U.S. Pat. No. 5,863,541.

[0165] Molecules that may be packaged by the inventive parvoviruscapsids and transferred into a cell include recombinant AAV genomes,which may advantageously may then integrate into the target cell genome,and other heterologous DNA molecules. RNA, proteins and peptides, orsmall organic molecules, or combinations of the same. Heterologousmolecules are defined as those that are not naturally found in anparvovirus infection, i.e., those not encoded by the parvovirus genome.In a preferred embodiment of the present invention, a DNA sequence to beencapsidated may be linked to an AAV ITR sequence that contains theviral packaging signals, which may increase the efficiency ofencapsidation and/or targeted integration into the genome.

[0166] The invention is further directed to the association oftherapeutically useful molecules with the outside of the inventiveparvovirus capsids for transfer of the molecules into host target cells.Such associated molecules may include DNA, RNA, carbohydrates, lipids,proteins or peptides. In one embodiment of the invention thetherapeutically useful molecules is covalently linked (i.e., conjugatedor chemically coupled) to the capsid proteins. Methods of covalentlylinking molecules are known by those skilled in the art.

[0167] The targeted and/or modified parvovirus capsid proteins, capsids,and virus particles of the invention find use for raising antibodiesagainst these novel capsid structures. Alternatively, an exogenous aminoacid sequence may be inserted into the parvovirus capsid for antigenpresentation to a cell, e.g. for administration to a subject to producean immune response to the exogenous amino acid sequence. According tothis lafter embodiment, it is not necessary that the exogenous aminoacid sequence also alter the tropism of the parvovirus.

[0168] It will be appreciated by those skilled in the art thatmodified/targeted viruses and capsids as described above may also bechimeric and/or hybrid parvoviruses as described in the precedingsections. Those skilled in the art will further appreciate that theinsertion mutants described herein include parvoviruses with othermodifications, e.g., deletion, insertion or missense mutations. Inaddition, the mutations may incidentally be introduced into theparvovirus capsid or rAAV genome as a result of the particular cloningstrategy employed.

[0169] Parvoviruses, AAV, and rAAV genomes are as described above withrespect to hybrid parvoviruses. The present invention also providescloning vectors, transcomplementing packaging vectors, packaging cells,and methods for producing the modified and/or targeted rAAV particlesdescribed above. In general, helpers, packaging cells, and methods forproducing the targeted or modified parvoviruses are as described abovewith respect to hybrid and chimeric viruses. In addition, at least oneof the cap genes (encoded by the rAAV genome, a packaging vector, or thepackaging cell) has inserted or substituted therein at least one nucleicacid sequence encoding an exogenous targeting sequence (as describedabove) or an exogenous amino acid sequence (as described above, e.g.,for purification, detection or antigen presentation).

[0170] IV. Gene Transfer Technology

[0171] The methods of the present invention provide a means fordelivering heterologous nucleic acid sequences into a broad range ofhost cells, including both dividing and non-dividing cells. The vectorsand other reagents, methods and pharmaceutical formulations of thepresent invention are additionally useful in a method of administering aprotein or peptide to a subject in need thereof, as a method oftreatment or otherwise. In this manner, the protein or peptide may thusbe produced in vivo in the subject. The subject may be in need of theprotein or peptide because the subject has a deficiency of the proteinor peptide, or because the production of the protein or peptide in thesubject may impart some therapeutic effect, as a method of treatment orotherwise, and as explained further below.

[0172] In general, the present invention may be employed to deliver anyforeign nucleic acid with a biological effect to treat or ameliorate thesymptoms associated with any disorder related to gene expression.Illustrative disease states include, but are not limited to: cysticfibrosis (and other diseases of the lung), hemophilia A, hemophilia B,thalassemia, anemia and other blood disorders, AIDs, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, epilepsy, and other neurological disorders, cancer, diabetesmellitus, muscular dystrophies (e.g., Duchenne, Becker), Gaucher'sdisease, Hurler's disease, adenosine deaminase deficiency, glycogenstorage diseases and other metabolic defects, retinal degenerativediseases (and other diseases of the eye), diseases of solid organs(e.g., brain, liver, kidney, heart), and the like.

[0173] Gene transfer has substantial potential use in understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Insome cases, the function of these cloned genes is known. In general, theabove disease states fall into two classes: deficiency states, usuallyof enzymes, which are generally inherited in a recessive manner, andunbalanced states, at least sometimes involving regulatory or structuralproteins, which are inherited in a dominant manner. For deficiency statediseases, gene transfer could be used to bring a normal gene intoaffected tissues for replacement therapy, as well as to create animalmodels for the disease using antisense mutations. For unbalanced diseasestates, gene transfer could be used to create a disease state in a modelsystem, which could then be used in efforts to counteract the diseasestate. Thus the methods of the present invention permit the treatment ofgenetic diseases. As used herein, a disease state is treated bypartially or wholly remedying the deficiency or imbalance that causesthe disease or makes it more severe. The use of site-specificintegration of nucleic sequences to cause mutations or to correctdefects is also possible.

[0174] The instant invention may also be employed to provide anantisense nucleic acid to a cell in vitro or in vivo. Expression of theantisense nucleic acid in the target cell diminishes expression of aparticular protein by the cell. Accordingly, antisense nucleic acids maybe administered to decrease expression of a particular protein in asubject in need thereof. Antisense nucleic acids may also beadministered to cells in vitro to regulate cell physiology, e.g., tooptimize cell or tissue culture systems. The present invention is alsouseful to deliver other non-translated RNAs, e.g., ribozymes (e.g., asdescribed in U.S. Pat. No. 5,877,022), RNAs that effectspliceosome-mediated trans-splicing (Puttaraju et al., (1999) NatureBiotech. 17:246), or “guide” RNAs (see, e.g., Gorman et al., (1998)Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan etal.) to a target cell.

[0175] Finally, the instant invention finds further use in diagnosticand screening methods, whereby a gene of interest is transiently orstably expressed in a cell culture system, or alternatively, atransgenic animal model.

[0176] V. Subjects, Pharmaceutical Formulations, Vaccines, and Modes ofAdministration

[0177] The present invention finds use in both veterinary and medicalapplications. Suitable subjects include both avians and mammals, withmammals being preferred. The term “avian” as used herein includes, butis not limited to, chickens, ducks, geese, quail, turkeys and pheasants.The term “mammal” as used herein includes, but is not limited to,humans, bovines, ovines, caprines, equines, felines, canines,lagomorphs, etc. Human subjects are the most preferred. Human subjectsinclude fetal, neonatal, infant, juvenile and adult subjects.

[0178] In particular embodiments, the present invention provides apharmaceutical composition comprising a virus particle of the inventionin a pharmaceutically-acceptable carrier or other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc. Forinjection, the carrier will typically be a liquid. For other methods ofadministration, the carrier may be either solid or liquid, such assterile, pyrogen-free water or sterile pyrogen-free phosphate-bufferedsaline solution. For inhalation administration, the carrier will berespirable, and will preferably be in solid or liquid particulate form.As an injection medium, it is preferred to use water that contains theadditives usual for injection solutions, such as stabilizing agents,salts or saline, and/or buffers.

[0179] In other embodiments, the present invention provides apharmaceutical composition comprising a cell in which an AAV provirus isintegrated into the genome in a pharmaceutically-acceptable carrier orother medicinal agents, pharmaceutical agents, carriers, adjuvants,diluents, etc.

[0180] By “pharmaceutically acceptable” it is meant a material that isnot biologically or otherwise undesirable, e.g., the material may beadministered to a subject without causing any undesirable biologicaleffects. Thus, such a pharmaceutical composition may be used, forexample, in transfection of a cell ex vivo or in administering a viralparticle or cell directly to a subject.

[0181] The parvovirus vectors of the invention maybe administered toelicit an immunogenic response (e.g., as a vaccine). Typically, vaccinesof the present invention comprise an immunogenic amount of infectiousvirus particles as disclosed herein in combination with apharmaceutically-acceptable carrier. An “immunogenic amount” is anamount of the infectious virus particles that is sufficient to evoke animmune response in the subject to which the pharmaceutical formulationis administered. Typically, an amount of about 10³ to about 10¹⁵ virusparticles, preferably about 10⁴ to about 10^(10,) and more preferablyabout 10⁴ to 10⁶ virus particles per dose is suitable, depending uponthe age and species of the subject being treated, and the immunogenagainst which the immune response is desired. Subjects and immunogensare as described above.

[0182] The present invention further provides a method of delivering anucleic acid to a cell. For in vitro methods, the virus -may beadministered to the cell by standard viral transduction methods, as areknown in the art. Preferably, the virus particles are added to the cellsat the appropriate multiplicity of infection according to standardtransduction methods appropriate for the particular target cells. Titersof virus to administer can vary, depending upon the target cell type andthe particular virus vector, and may be determined by those of skill inthe art without undue experimentation. Alternatively, administration ofa parvovirus vector of the present invention can be accomplished by anyother means known in the art.

[0183] Recombinant virus vectors are preferably administered to the cellin a biologically-effective amount. A “biologically-effective” amount ofthe virus vector is an amount that is sufficient to result in infection(or transduction) and expression of the heterologous nucleic acidsequence in the cell. If the virus is administered to a cell in vivo(e.g., the virus is administered to a subject as described below), a“biologically-effective” amount of the virus vector is an amount that issufficient to result in transduction and expression of the heterologousnucleic acid sequence in a target cell.

[0184] The cell to be administered the inventive virus vector may be ofany type, including but not limited to neural cells (including cells ofthe peripheral and central nervous systems, in particular, brain cells),lung cells, retinal cells, epithelial cells (e.g., gut and respiratoryepithelial cells), muscle cells, pancreatic cells (including isletcells), hepatic cells, myocardial cells, bone cells (e.g., bone marrowstem cells), hematopoietic stem cells, spleen cells, keratinocytes,fibroblasts, endothelial cells, prostate cells, germ cells, and thelike. Alternatively, the cell may be any progenitor cell. As a furtheralternative, the cell can be a stem cell (e.g., neural stem cell, liverstem cell). Moreover, the cells can be from any species of origin, asindicated above.

[0185] In particular embodiments of the invention, cells are removedfrom a subject, the parvovirus vector is introduced therein, and thecells are then replaced back into the subject. Methods of removing cellsfrom subject for treatment ex vivo, followed by introduction back intothe subject are known in the art. Alternatively, the rAAV vector isintroduced into cells from another subject, into cultured cells, or intocells from any other suitable source, and the cells are administered toa subject in need thereof.

[0186] Suitable cells for ex vivo gene therapy include, but are notlimited to, liver cells, neural cells (including cells of the centraland peripheral nervous systems, in particular, brain cells), pancreascells, spleen cells, fibroblasts (e.g., skin fibroblasts),keratinocytes, endothelial cells, epithelial cells, myoblasts,hematopoietic cells, bone marrow stromal cells, progenitor cells, andstem cells.

[0187] Dosages of the cells to administer to a subject will vary uponthe age, condition and species of the subject, the type of cell, thenucleic acid being expressed by the cell, the mode of administration,and the like. Typically, at least about 10² to about 10^(8,) preferablyabout 10³ to about 10⁶ cells, will be administered per dose. Preferably,the cells will be administered in a “therapeutically-effective amount”.

[0188] A “therapeutically-effective” amount as used herein is an amountof that is sufficient to alleviate (e.g., mitigate, decrease, reduce) atleast one of the symptoms associated with a disease state. Alternativelystated, a “therapeutically-effective” amount is an amount that issufficient to provide some improvement in the condition of the subject.

[0189] A further aspect of the invention is a method of treatingsubjects in vivo with the inventive virus particles. Administration ofthe parvovirus particles of the present invention to a human subject oran animal in need thereof can be by any means known in the art foradministering virus vectors.

[0190] Exemplary modes of administration include oral, rectal,transmucosal, topical, transdermal, inhalation, parenteral (e.g.,intravenous, subcutaneous, intradermal, intramuscular, andintraarticular) administration, and the like, as well as direct tissueor organ injection, alternatively, intrathecal, direct intramuscular,intraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Injectables can be prepared in conventionalforms, either as liquid solutions or supenisions, soild forms suitablefor solution or suspenions in liquid prior to injection, or asemulsions. Alternatively, one may administer the virus in a local ratherthan systemic manner, for example in a depot or sustained-releaseformation.

[0191] In particurly preformed embodiments of the invention, thenucleotide sequence of intrest is delivered to the liver of the subject.Adminstration to the liver may be achieved by any method known in art,including, but not limited to intravenous administration, intraportaladministration, intrabilary administration, intra-arterialadministration, and direct injection into the liver paraenchyma.

[0192] Preferably, the cells (e.g., liver cells) are infected by arecombiant parvovirus vector encoding a peptide or protein, the cellsexpress the encoded peptide or protein and secrete it into thecirculatory system in a therapeutically-effective amount (as definedabove). Alternatively, the vector is delivered to and expressed byanother cell or tissue, including but not limited to, brain, pancreas,spleen or muscle.

[0193] In other preferred embodiments, the inventive parovirus particlesare administered intramuscularly, more preferably by intramuscularinjection or by local administration (as defined above). In otherpreferred embodiments, the parovirus particles of the present inventionare administered to the lungs.

[0194] The parovirus vector disclosed herein may be administered to thelungs of a subject by any suitable means, but are preferablyadministered by adminsitering an aresol suspension of respirableparticles comprised of the inventive parovirus vectors, which thesubject inhales. The respirable particles may be liquid or solid.Aerosols of liquid particles comprising the inventive parovirus vectorsmay be produced by any suitable means, such as with a pressure-drivenaerosol nebulizer or an ultrasonic nebulizer, as is known to those ofskill in art. See, e.g. U.S. Pat. No. 4,501,729. Aerosols of solidparticles comprising the inventive virus vectors may likewise beproduced with any solid particulate medicament aerosol generator, bytechniques known in the pharmaceutical art.

[0195] Dosages of the inventive parvovirus particles will depend uponthe mode of administration, the disease or condition to be treated, theindividual subject's condition, the particular virus vector, and thegene to be delivered and can be determined in a routine manner.Exemplary doses for achieving therapeutic effects are virus titers of atleast about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵transducting units or more, preferably about 10⁸-10¹³ transductingunits, yet more preferably 10¹² transducing units.

[0196] In particular embodiments of the invention, more than oneadministration (e.g., two, three, four, or more administrations) may beemployed to achieve therapeutic levels of gene expression. According tothis embodiment and as described above, it is preferred to useparvovirus vectors having different entigenic properties for eachadministration to obviate the effects of neutralizing antibodies. Asdescribed above, in particular embodiments of the invention, the hybridand chimeric parvoviruses of the present invention are administered tocircumvent neutralizing antibodies in the subject to be treated or toprevent the development of an immune response in the subject. Thesubject may be presented with seemingly new virus vectors by packagingthe rAAV genome within an array of hybrid or chimeric parvoviruscapsids.

[0197] The foregoing discussion also pertains to pharmaceuticalformulations containing parvovirus capsids and other reagents of theinvention as well as methods of administering the same.

[0198] In summary, the parvovirus vectors, reagents, and methods of thepresent invention can be used to direct a nucleic acid to eitherdividing or non-dividing cells, and to stably express the heterologousnucleic acid therin. Using this vector system, it is now possible tointroduce into cells, in vitro or in vivo, genes that encode proteinsthat affect cell physiology. The vectors of the present invention canthus be useful in gene therapy for disease states or for experimentalmodification of cell physiology.

[0199] Having now described the invention, the same will be illustratedwith reference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

EXAMPLE 1 AAV Vectors

[0200] All production of AAV vectors used in these investigationsutilized the vector production scheme as described in Ferrari et al.,(1997) Nature Med. 3:1295 and Xiao et al., (1998) J. Virology 72:2224.Utilizing a transient transfection procedure, rAAV devoid of adenovirushas been generated. Id. This protocol utilizes an adenovirus DNA genomethat has been incapacitated for viral replication and late geneexpression. The mini Ad plasmid while unable to replicate and produceprogeny, is still viable for adenovirus gene expression in 293 cells.Using this construct, the AAV packaging strategy involving new AAVhelper plasmid (pAAV/Ad ACG) and AAV vector DNA (sub 201) has beensuccessfully complemented (Samulski et al., (1989) J of Virology63:3822). This new construct typically generates rAAV of 10⁷-10⁹/10 cmdish of 293 cells (Xiao et al., (1998) J. Virology, 72:2224). Efficientgene delivery is observed in muscle, brain and liver with these vectorsin the complete absence of Ad.

EXAMPLE 2 Cells and Viruses

[0201] Human 293 and HeLa cells were maintained at 37° C. with 5%CO₂saturation in 10% fetal bovine serum (Hyclone) in Dulbecco's modifiedEagles medium (Gibco BRL), with streptomycin and penicillin (LinebergerComprehensive Cancer Center, Chapel Hill, N.C.) Four×10⁶293 cells wereplated the day before transfection onto a 10 cm plate. Cells weretransfected by both calcium phosphate (Gibco BRL) or Superfection(Qiagene) according to manufacturers specifications. The insertionalmutant packaging plasmids, described below, were transfected along withpAB11 containing the CMV driven Lac Z gene with a nuclear localizationsignal. For each transfection the same amount of packaging plasmid (12μg) and pAB11 (8 μg) were used for each 10 cm plate. For eachtransfection an additional plate was used containing the transgeneplasmid only to assess transformation efficiencies. After transfectionthe cells were infected with helper virus Ad5 dl309 at an MOI of 5, and48 hours later the cells were lysed and the virus purified.

[0202] Recombinant virus was purified using cesium chloride isopycnic oriodixanol gradients. In both cases cells were centrifuged at 1500 rpms(Sorvall RT 6000B) for ten minutes at 4° C. Proteins were precipitatedfrom the supernatant using ammonium sulfate (30% w/v) and resuspended in1×Phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mMNa₂HPO₄7H₂O, 1.4 mM KH₂PO₄). The cell pellet was resuspended in 1×PBScontaining 0.1 mg/ml DNase I (Boehringer Mannheim) lysed by threefreeze-thaw cycles, combined with the protein portion of thesupernatant, and incubated at 37° C. for 30 minutes. This material wassubjected to sonication (Branson Sonifer 250, VWR Scientific), 25 burstsat 50% duty, output control 2. Cell debris was removed by centrifugation(Sorvall RT 6000B). To each milliliter of supernatant 0.6 g of cesiumchloride (CsCl) was added and the solution was centrifuged for 12-18hours (Beckman Optima TLX ultracentrifuge) in a TLS 55 rotor at 55,000rpms. Alternatively, the supernatant was layered on top of an Iodixanol(OptiPrep -Nycomed Pharma As, Oslo, Norway) gradient of 60%, 45%, 30%and 15%. This gradient was centrifuged in a Beckman Optima TLXultracentrifuge using a TLN 100 rotor at 100,000 rpm for one hour.Fractions were recovered from these gradients and 10 μl from eachfraction were utilized for dot blot hybridization to determine whichfraction contained the peack protected viron (see Example 5).

EXAMPLE 3 Construction of AAV Packaging Plasmids

[0203] The capsid domain of pAAV/Ad was cloned into pBS×(Stratagene)using Hind III, resulting in pAV2Cap. Partial digestion of pAV2Cap usingthe restriction enzymes Hae III, Nla IV, and Rsa I and gel purificationof the unit length DNA fragment resulted in the isolation of thestarting material for cloning. The aminoglycoside 3′-phosphotranferasegene, conferring kanamycin resistance (kan'), from pUC4K (Pharmacia)digested with Sal I was flanked by linkers containing Nae I and Eco RVsites, a Sal I overhang at one end and an Eco RI overhang at the otherend (top 5′-AATTCGCCGGCGATATC-3′, SEQ ID NO:6, bottom5′-TCGAGATATCGCCGGC-3′SEQ ID NO:7). This fragment was cloned into theEco RI site of pBluescript SK×(Stratagene). Digestion with Nae Ireleased the kan^(r)gene, and this fragment was ligated into the pAV2Cappartials. The resulting plasmids were screened for insertion into thecapsid domain and, then digested with Eco RV to remove the kan^(r)geneleaving the twelve base pair insertion 5′-GGCGATATCGCC-3′(SEQ ID NO: 8)within the capsid domain. Multiple enzyme digests and DNA sequencingwere used to determine the position of the 12 bp insertion within thecapsid coding domain. The enzyme digests include Eco RV/Ban II, EcoRV/Bst NI, Eco RV/Pst I/ and Eco RV/Hind III. The capsid domain of theresulting plasmids were digested with Asp718 and subcloned into thepACG2 packaging plasmid (Li et al., 1997 J. Virology 71:5236), with theexception of one NlaIV clone that overlapped the 3′-Asp718 site. Thisinsertion mutant was cloned into pAAV/Ad using a Hind III/Nsi Idigestion.

EXAMPLE 4 Western Blotting

[0204] Cell lysates after freeze thaw lysis and sonication wascentrifuged to remove large cell debris. Twenty microliters ofsupernatant was immediately added to 20 μl of 2×SDS gel-loading buffercontaining dithiothreitol and boiled for five minutes. Proteins wereanalyzed by SDS polyacrylamide gel electrophoresis and transferred tonitrocellulose electrophoretically. The nitrocellulose membranes wereimmunoblotted using the anti-Vp3 monoclonal antibody B1 (a generous giftfrom Jurgen A. Kleinschmidt). Each of the insertion mutants was testedat least twice by Western blot analysis. The secondary anti-mouseHorseradish Peroxidase IgG was used to indirectly visualize the proteinby enhanced chemiluminescence (ECL-Amersham). The Western blots werescanning from enhanced chemiluminescence exposed BioMax film (Kodak)into Adobe PhotoShop and analyzed by ImageQuaNT software (MolecularDynamics Inc.).

[0205] Viral proteins were visualized by Western blotting followed byimmunoblotting as described above. Between 1.0×10⁹ and 2.5×10⁹ viralparticles were used for each sample. The virus was isolated from thepeak cesium gradient fraction as determined by dot blot, and dialysedagainst 0.5× PBS containing 0.5 mM MgCl₂ prior to polyacrylamide gelelectophoresis.

EXAMPLE 5 Titration of Recombinant Virus

[0206] Fractions from CsCl gradients were obtained by needle aspiration.The refractive index was obtained using a refractometer (Leica Mark II),and the index was used to determine the density of fractions. Aliquotsof 10 μl from fractions between 1.36 g/ml and 1.45 g/ml were tested forthe presence of protected particles by dot blot hybridization. Thealiquots were diluted 1:40 in viral dilution buffer (50 mM Tris HCl, 1mM MgCl₂, 1 mM CaCl₂ 10 μg/ml RNase, 10 μg/ml DNase) and incubated at37° C. for 30 minutes. To the samples Sarcosine (final concentration0.5%) and EDTA (final 10 mM) were added and incubated at 70° C. for 10minutes. Proteinase K (Boehringer Mannheim) was added to a finalconcentration of 1 mg/ml and the samples were incubated at 37 ° C. fortwo hours. Following this incubation the samples were denatured in NaOH(350 mM final) and EDTA (25 mM final). The samples were applied toequilibrated nytran (Gene Screen Plus, NEN Life Science Products) usinga dot blot manifold (Minifold I, Schleicher and Schuell). The membranewas probed with a random primed (Boehringer Mannheim) ³²P-dCTP labeledLac Z DNA fragment. The membranes were exposed to film (BioMax MR,Kodak) or to phosphor imagining screens (Molecular Dynamics) andintensity estimates were done using ImageQuant software (MolecularDynamics). Peak fraction of virus were then dialysed in 1×PBS fortransducing filter.

[0207] Transductions titers were determined by histochemical stainingfor Lac Z activity. HeLa cells had been infected with Ad dl309 at amultiplicity of infection of five for one hour. The cells were thenwashed with 1×PBS and fresh medium was added. Aliquots of virus frompeak fractions, equivalent to 1.75×10⁸ particles were used to infectHela cells. Twenty to twenty-four hours later cells were washed with1×PBS, fixed (2% formaldehyde 0.2% gluteraldehyde in 1×PBS), washed, andstained with 5′-Bromo-4-chloro-3-indoly-β-D-galactophyranoside (Gold BioTechnology) dissolved in N,N-dimethylformamide (Sigma) diluted to 1mg/ml in 1×PBS pH7.8, 5mM potassium ferricyanide, 5 mM potassiumferrocyanide, 2 mM MgCl2 at 37° C. for 12-24 hours. Stained HeLa cellswere counted in ten 400× microscope fields. The transducing number wasdetermined by averaging the number of stained cells in ten fields andmultiplying by the number of fields on the plate and dividing thatnumber by the number of nanograms of protected template.

EXAMPLE 6 Electron Microscopy

[0208] Peak fractions of rAAV with wildtype viron or mutagenized virionswere dialysed in 0.5×PBS containing 0.5 mM MgCl₂. The virus was placedon a 400 mesh glow dischared carbon grid by inverting on a 10 μl drop ofvirus for ten minutes at room temperature. Followed by three 1×PBSwashes for one minute each. The virus was stained in 1% Phosphotungsticacid for one minute. Specimens were visualized using a Zeiss EM 910electron microscope.

EXAMPLE 7 Heparin Agarose Binding Assay

[0209] Recombinant virus containing wild-type capsids or insertion inthe capsids were dialysed against 0.5×PBS containing 0.5 mM MgCl₂. Onehundred microliters of each virus was bound to 100 μl of heparin agarosetype 1 (H-6508 Sigma, preequilibrated in twenty volumes of 0.5×PBScontaining 0.5 mM MgCl₂) at room temperature for one hour in a 1.5 mlmicrofuge tube. After each step, binding washes and elutions sampleswere centrifuged at 2000 rpm (Sorvall MC 12V) for two minutes to collectsupernatant. Samples were washed six times with 0.5 ml of 0.5×PBScontaining 0.5 mM MgCl₂, and the supernatant collected. Samples wereeluted in three steps of 100 μl volumes containing 0.5, 1.0 and 1.5 MNaCl in 0.5×PBS containing 0.5 mM MgCl₂ and the supernatant collected.For each sample 20 μl of supernatant from each step was used for dotblot hybridization. The 100% bound control was an internal standardequivalent to one fifth of each input virus used in the dot blot. Theheparin agarose viral mixtures were washed six times with 0.5×PBS 0.5 mMMgCl₂ in volumes that resulted in a 1:15625 dilution.

EXAMPLE 8 Construction of Insertional Mutations in rAAV2

[0210] In order to evaluate the role of AAV structural proteins inassembly and infectivity, we generated a collection of capsid linkerinsertion mutants. A 2.8 kb Hind III fragment of pAAV/Ad (Samulski etal., (1989) J. Virology 63:3822) containing the sequences coding for thecapsid domain of AAV2 was subcloned into pBS+. This plasmid, pAV2Cap,was used for partial digestion with Hae III, Nla IV, and Rsa I togenerate a substrate for capsid specific insertions (FIG. 1). Thesethree DNA restriction enzymes constitute 43 sites that span across theAAV-2 capsid coding sequence of which only 4 overlap. To efficientlyidentify clones that contain insertions, a kanamycin resistance gene(Kan^(r)) flanked by a novel oligo (Nae I/EcoR V) was ligated withpartially digested, full-length, linearized pAV2Cap (see Example 3 andFIG. 1). Using ampicillin and kanamycin selection in E. coli, insertionmutants were identified and the Kan^(r)gene was shuttled out of thecapsid coding region by digesting and religation with the nested pair ofEco RV sites (see Example 3). This resulted in a specific linkerinsertion of 12 base pair (bp) carrying a single copy of the unique EcoRV site in the capsid coding sequences. The exact positions of thelinker insertion were further refined by restriction enzyme digestions,and in six cases sequencing (data not shown). The position of insertionmutants are identified by the first letter of the enzyme used in thepartial digestion followed by the nucleotide position of the restrictionsite in the AAV2 genome, for example Nla IV 4160 would be N4160.

[0211] The capsid coding sequence from these mapped insertion mutantswere subcloned into the helper vectors pACG2 or pAAV/Ad for biologicalcharacterization in vivo (FIG. 1) (Li et al., (1997) J. Virology71:5236; Samulski et al., (1989) J. Virology 63:3822). Sequence analysispredicts that this 12 base pair insertion cannot result in a terminationcodon for any of the 43 insertion sites (Table 1). Owing to the randomnature of the cut site for the enzymes (Hae III, Nla IV, and Rsa I) withrespect to codon frame usage and the degeneracy of the Nla IVrecognition sequence, the 12 bp linker resulted in the insertion of theamino acids GDIA in frame 1 and AISP in frame 3 for all three enzymes,while insertions in frame 2 resulted in WRYRH for Rsa I. GRYRP for HaeIII, and both GRYRP and GRYRH for Nla IV. The bolded amino acid in theseexamples represents missense mutation (Table 1). The mutant helperconstructs, pACG2^(IN,) were individually transfected into 293 cellsalong with an AAV reporter vector, containing the β-galactosidase genein Adenovirus dl309 (MOI=5) infected cells (li et al., (1997) J.Virology 71:5236). The transfected cells were then assayed for capsidexpression and recombinant virus production (see Example 5; Li et al.,(1997) J. Virology 71:5236). TABLE 1 Physical Structure and Phenotype ofAAV2 Capsid Insertion Mutants Position¹ Capsid Heparin Electron insertedsubunit Frame² Dot blot³ Infectious⁴ Agarose⁵ Microscope Phenotype AminoAcid⁶ H2285 VP1 3 2.8 × 10⁷ − + normal Class II AISP R2356 VP1 2 1.4 ×10⁸ + + N.D. Class III WRYRH N2364 VP1 1 — − N.D. N.D. Class I GDIAH2416 VP1 2 1.4 × 10⁷ − + N.D. Class II GRYRP H2591 VP1 3 1.4 × 10⁷ + +normal Class III AISP H2634 VP2 1 2.8 × 10⁷ − + normal Class II GDIAH2690 VP2 3 7.0 × 10⁶ + + normal Class III AISP R2747 VP2 3 — − N.D.N.D. Class I AISP H/N2944 VP3 2 1.4 × 10⁶ +* N.D. N.D. Class II/IIIGRYRP N3317 VP3 3 1.4 × 10⁵ − N.D. N.D. Class II AISP R3391 VP3 2 — −N.D. N.D. Class I WRYRH N3561 VP3 1 — − N.D. N.D. Class I GDIA H3595 VP32 1.4 × 10⁶ +* N.D. abnormal Class II/III GRYRP H/N3761 VP3 3 1.4 × 10⁷− − normal Class II AISP H3766 VP3 2 2.8 × 10⁷ − N.D. N.D. Class IIGRYRP N4046 VP3 3 — − N.D. N.D. Class I AISP H/N4047 VP3 1 — − N.D. N.D.Class I GDIA N/R4160 VP3 3 1.4 × 10⁷ + + normal Class III AISP

EXAMPLE 9 Analysis of Capsid Proteins

[0212] Before assaying for vector production using mutant capsidconstructs in complementation assays, each insertion mutant was testedfor expression of capsid subunits in 293 cells after transfection. Theability to produce Vp1, Vp2, and Vp3 at normal stoichiometry wouldsuggest that linker insertions did not alter capsid protein expression,or stability. Since the linker did not introduce stop codons, it wasexpected that each insert would produce all three capsids. Forty-eighthours after transfection, cell lysates were analyzed by Western blot forAAV capsids. The Western blot analysis in FIG. 2 is a representation ofinsertion mutant capsid expression in cell lysates. With the exceptionof H2634 (FIG. 2 lane 2), the stoichiometry of the three capsid subunitsdoes not appear significantly different than that of wild-type controls(FIG. 2 compare lanes 1,3-7 to lane 8). By this assay, insertion mutantH2634 appears to only produce Vp3 subunits (FIG. 2; lane 2). In longerexposures, the minor capsid subunits in FIG. 4 lanes 4 and 5 wereapparent (data not shown).

EXAMPLE 10 Mutant Capsid Ability to Produce Stable Virions

[0213] To test for the production of stable virions that protect avector genome from DNase digestion, we subjected the cell lysates tocesium chloride (CsCl) gradient centrifugation. Virus densities weremeasured by refractometry, and aliquots from appropriate fractions weresubjected to dot blot hybridization (FIG. 3a). Based on this analysis,particles that package intact recombinant genomes should display abuoyant density similar to wild-type and be resistant to DNasetreatment, with the exception of H2944 which has a buoyant densityslightly higher than wild type. Results for this assay separatedinsertion mutants into two classes. Class I mutants were negative forprotecting the viral genome, while class II mutants appeared normal forpackaging and protecting the vector substrate (Table I).

[0214] All class II mutants had a buoyant density within the range ofwild-type AAV2 capsids (FIG. 3a). By dot blot analysis, N2944 packagedthe recombinant genome but migrated to a position of slightly greaterdensity than wild type in isopycnic gradients (FIG. 3a, N2944 lane 3). Anumber of insertion mutants (7) did not package DNA by this assay whichhad a sensitivity of <1×10⁵ particles/μl (see methods for quantitation)(Table 1). Whether these mutants were defective in packaging or unstableduring purification remains to be determined.

EXAMPLE 11 Infectivity of Class II Insertion Mutants

[0215] Virions generated by insertion mutants in the complementationassay were tested for infectivity by monitoring transduction of LacZreporter gene in human cells. Using viral titers derived from dot blothybridization, HeLa cells were infected with mutant virus stocks atequivalent particle numbers.

[0216] Twenty-four hour post infection, expression of the transgene wasdetected by X-gal staining. A representative figure of this analysis isshown (FIG. 3b) and all mutanta assayed are presented in Table 1. Inthis assay, wild-type virions transduced 5.6×10⁵ HeLa cells/1.75×10⁸protected particles (FIG. 3b). Based on the sensivity of this assay, therange of infection efficiency for class II insertion mutant viruses wasfrom 0 to 1.6×10⁶ transducing units/1.75×10⁸ protected particles.Results from this analysis further subdivided the capsid insertionmutants from class II (normal for packaging and protecting the vectorsubstrate) into a class III phenotype (normal for packaging andprotecting the vector substrate and infectious virions). Two insertionmutants negative for infectivity and initially identified as class IImutants (N2944, H3595) based on CsCl purification and DNase protection,tested positive for viral transduction after purification using aniodixanol step gradient (Table 1). This virus purification technique isnot as harsh as CsCl and has been shown to increase virus recovery byten-fold (Zolotukhin et al., (1999) Gene Therapy 6:973). However, otherclass II mutants remained non-infectious after purification using aniodixanol step gradient (data not shown). Although we determined thatinsertion mutant viruses N2944 and H3595 were infectious using the Lac Ztransduction assay, it should be noted that these mutants resulted inlow infectious titers (1×10² transducing units/ng) similar to previouslypublished lip mutants (Hermonat et al., (1984) J. Virology 51:329).

EXAMPLE 12 Electron Microscopy of Class II and Class III Mutants

[0217] To further characterize class II and III rAAV2 insertion mutantsfor biological differences, we visualized mutant particles by electronmicroscopy (EM). The EM analysis revealed only gross morphology of theinfectious class III viruses, which were indistinguishable fromwild-type virions (Compare FIG. 4a, and 4 b,c). Whereas distinctdifferences were observed between class II/III mutant virus H3595 whencompared to wild-type virions (FIG. 4a, and 4 f-bottom four panels). EMimages of H3595 revealed a slightly larger roughly pentagonal outline,while wild-type virus appeared uniformed in size and was hexagonal.Interestingly, class II mutant H2634, which was negative for Vp1 or Vp2by Western blot (FIG. 2 lane 2), appeared normal in morphology by EManalysis (FIG. 4d). Based on this analysis, virion morphology alone isnot sufficient to distinguish class II mutants from class III sincesmall insertions within the capsids can result in either non-detectable(FIG. 4b,c,d,e) or noticeable alterations in virion structure (FIG.4f-bottom four panels). However, this approach was able to provideadditional data to our characterization of these linker insertionmutants (FIG. 4, compare a to f).

EXAMPLE 13 Capsid Ratio of Class II and Class III Virions

[0218] Rose et al., (1971) established that AAV2 particles are composedof Vp1, Vp2, and Vp3 at a 1:1:20 ratio (Rose et al., (1971) J. Virology8:766). In an effort to determine if class II and class III mutantvirions maintained this ratio, Western blots were performed on thecesium chloride purified virus. Purified viruses analyzed by Westernblot showed similar amounts of Vp3 in all mutants sampled (FIG. 5, Vp3arrow), between 1×10⁹ and 2.5×10⁹ viral particles were used for eachsample. The amounts of Vp2 and Vp1 are also nearly equivalent in alltest samples except H2634 where no minor capsid components were observed(FIG. 5, lane 5). The lack of minor capsid components for H2634 isconsistent with the Western results from cell lysate (FIG. 2). At thelimit of detection in this assay, the class II insertion mutant H2634appears to assemble AAV virions without Vp1 and Vp2, even though EManalysis suggest this mutant has normal morphology (FIG. 4d).

EXAMPLE 14 Heparin Binding of Class II and Class III Mutants

[0219] Recently our lab established that AAV-2 uses a heparan sulfateproteoglycan as a primary receptor for infectivity (Summerford andSamulski, (1998) J. Virology 72:1438). To determine what role heparinbinding may have in class II particles inability to infect cells as wellas the ability of class III virus to bind heparin agarose, heparin batchbinding experiments were performed. Not surprisingly, all class IIImutants were positive for heparin binding, with the majority of viruseluting in the 1M NaCl₂ step (data not shown). To determine if loss ofinfectivity of class II mutant viruses was related to a lack of heparinbinding, batch binding experiments were analyzed by dot blothybridization (FIG. 6). For each of the viral samples tested, aninternal control to determine 100% bound was spotted on the filterindependent of heparin binding (FIG. 6; 100% bound). This allowed us todetermine percent virus retained, at each step of heparin purification.After binding to heparin agarose, samples were washed then eluted usingincreasing salt concentrations (see Example 7). Recombinant AAV2 withwild-type virion shells demonstrated 90% binding with 10% released inthe wash followed by 60% recovered in the elution buffer, and 20%remaining bound to heparin agarose (FIG. 6, lane 1). Class II mutantsH2285, H2416, and H2634 demonstrated similar binding and elutionprofiles (FIG. 6, lanes 2-4). However, class II mutant H3761 wasdistinct in its heparin agarose binding profile with the majority of thevirion in the binding buffer and the washes (FIG. 6, lane 5). Furtheranalysis is required to determine the reason for lack of Heparin bindingin this batch assay.

[0220] Interestingly, H2634 binds heparin agarose under theseconditions, which by Western blot does not carry detectable Vp1 or Vp2subunits (FIG. 5, lane 4). The lack of Vp1 and Vp2 in H2634 along withits ability to bind heparin agarose suggest that the heparin bindingdomain may be located in Vp3 capsid proteins.

EXAMPLE 15 Linker Insertion Mutants

[0221] Insertion sequences encoding poly-lysine, poly-histidine, an RGDmotif, or bradykinin were inserted into the linker mutants described inTable 1. We developed a PCR-based method of identifying insertions ofdifferent linkers into the coding domain of AAV2 capsid gene. Briefly,one primer was used outside of the capsid coding region and one thatcorresponds exactly to the linker. If the linker is in the correctorientation, then the PCR product is of a size that is dependent on theinsertion mutant's positon.

[0222] After transformation of the ligation reactions, bacterialcolonies were picked with a pipet tip and dipped 4-5 times into a wellof a 96-well plate containing LB-medium with antibiotic. The pipet tipwas then placed in a well of a 96-well plate containing PCR reactionbuffer. The PCR products were run out on an agarose gel, and positiveclones were identified. This information indicated the orientation andthe position of the insertion mutant with respect to the outside primer.

[0223] The LB-medium that is in the corresponding well was used as thePCR positives, and this material was grown in a larger (5 mL) volume.After an overnight growth phase, the plasmid DNA was islolated anddigested with an enzyme that restricts the DNA 15 times (Bst NI). Thesedigestion products were separated on a 5-6% acrylamide gel. Dependingupon the size of the linker insertion and the size of the correspondinguninserted fragment, the number of inserts is determined. This, withintwo days of ligating the linker into the insertion site, we know theorientation and number of linker insertion, and we have sufficient DNAto transfect a 10 cm plate for virus production.

[0224] pACG2 (Li et al., 1997 J. Virology 71:5236) without any insertionwhen digested with Bst N1 yields fragments of:

[0225] 3900 bp

[0226] 1121 bp

[0227] 1112 bp

[0228] 445 bp—H2944 shifts

[0229] 347 bp—H2634, H2690 shifts

[0230] 253 bp—H3595 shifts

[0231] 215 bp—R2356, H2416 shifts

[0232] 121 bp

[0233] 111 bp

[0234] 64 bp

[0235] 63 bp—H2285 shifts

[0236] 33 bp—H2591 shifts

[0237] 13 bp

[0238] 9 bp

[0239] The band shifts with the different insertion mutants are alsoindicated.

[0240] pACG2 without any insertion when digested with Ban I yieldsfragments of:

[0241] 2009 bp

[0242] 1421 bp

[0243] 168 bp

[0244] 843 bp—H4047 shifts

[0245] 835 bp

[0246] 734 bp—H2634 shifts

[0247] 464 bp

[0248] 223 bp

[0249] 218 bp

[0250] 211 bp

[0251] 50 bp

[0252] Each of the inserts contains the original 12 base pairs of theEco RV site. In addition, each of the linkers adds additional basepairs:

[0253] RGD=36 bp+12=48 bp for a single insertion.

[0254] Bradykinin (BRDY)=69 bp+12=81 bp for a single insertion. Note:The BRDY insert contains a BstNI site.

[0255] Histidine (8HIS)=51 bp+12=63 bp for a single insertion.

[0256] Poly Lysine (PLY)=63 bp+12=75 bp for a single insertion.

[0257] The outside primer is near the Hind III site and is called AAV2/45′. This primer can be used to amplify AAV serotypes 2 and 4.

[0258] Primer sequences used to produce epitope linkers into theoriginal insertion mutants are given below. Note: Because there arethree frames for the insertion mutants there are three primer pairs foreach primer set.

[0259] Histidine primer pairs: Frame 1: Top primer a 48mer: 5′-GCT AGCGGC GGA CAC CAT CAC CAC (SEQ ID NO:9) CAC CAT CAC CAC GGC GGA AGC GCT-3′Bottom primer a 48mer: 5′-AGC GCT TCC GCC GTG GTG ATG GTG (SEQ ID NO:10)GTG GTG ATG GTG TCC GCC GCT AGC-3′ Frame 2: Top primer a 51mer: 5′-ACGCT AGC GGC GGA CAC CAT CAC (SEQ ID NO:11) CAC CAC CAT CAC CAC GGC GGAAGC GCT T-3′ Bottom primer a 51mer: 5′-A AGC GCT TCC GCC GTG GTG ATG(SEQ ID NO:12) GTG GTG GTG ATG GTG TCC GCC GCT AGC GT-3′ Frame 3: Topprimer a 51mer: 5′-G GGT TCC GGA GGG CAC CAC CAT (SEQ ID NO:13) CAC CACCAC CAT CAC GGA GGC GCC AGC GA-3′ Bottom primer a 51mer: 5′-TC GCT GGCGCC TCC GTG ATG GTG (SEQ ID NO:14) GTG GTG ATG GTG GTG CCC TCC GGA ACCC-3′ Bradykinin primer pairs: Frame 1: Top primer a 60mer: 5′-GCC GGATCC GGC GGC GGC TCC AGA (SEQ ID NO:15) CCC CCC GGC TTC AGC CCC TTC AGATCC GGC GGC GCC-3′ Bottom primer a 60mer: 5′-GGC GCC GCC GGA TCT GAA GGGGCT (SEQ ID NO:16) GAA GCC GGG GGG TCT GGA GCC GCC GCC GGA TCC GGC-3′Frame 2: Top primer a 69mer: 5′-GA GGT TCA TGT GAC TGC GGG GGA (SEQ IDNO:17) AGA CCC CCT GGC TTC AGC CCA TTC AGA GGT GGC TGC TTC TGT GGC G-3′Bottom primer a 69mer: 5′-C GCC ACA GAA GCA GCC ACC TCT (SEQ ID NO:18)GAA TGG GCT GAA GCC AGG GGG TCT TCC CCC GCA GTC ACA TGA ACC TC-3′ Frame3: Top primer a 60mer: 5′-A GGT TCA TGT GAC TGC GGG GGA (SEQ ID NO:19)AGA CCC CCT GGC TTC AGC CCA TTC AGA GGT GGC TGC TTC TGT GGC GG-3′ Bottomprimer a 60mer: 5′-CC GCC AGA GAA GCA GCC ACC TCT (SEQ ID NO:20) GAA TGGGCT GAA GCC AGG GGG TCT TCC CCC GCA GTC ACA TGA ACC T-3′ RGD primerpairs: Frame 1: Top primer a 36mer: 5′-GGA TCC TGC GAC TGC AGG GGC GAT(SEQ ID NO:21) TGT TTC TGC GGC-3′ Bottom primer a 36mer: 5′-GCC GCA GAAACA ATC GCC CCT GCA (SEQ ID NO:22) GTC GCA GGA TCC-3′ Frame 2: Topprimer a 36mer: 5′-GA TCC TCG GAC TGC AGG GGC GAT (SEQ ID NO:23) TGT TTCTGC GGC G-3′ Bottom primer a 36mer: 5′-C GCC GCA GAA ACA ATC GCC CCT(SEQ ID NO:24) GCA GTC GCA GGA TC-3′ Frame 3: Top primer a 36mer: 5′-AGGA TCC TGC GAC TGC AGG GGC (SEQ ID NO:25) GAT TGT TTC TGC GG-3′ Bottomprimer a 36mer: 5′-CC GCA GAA ACA ATC GCC CCT GCA (SEQ ID NO:26) GTC GCAGGA TCC T-3′ Polylysine primer pair: Note: only the frame three primerpair was made. Frame 3: Top primer a 63mer: 5′-A GGT TCA TGT GAC TGC GGGGGA (SEQ ID NO:27) AAG AAG AAG AAG AAG AAG AAG GGC GGC TGC TTC TGT GGCGG-3′ Bottom primer a 63mer: 5′-CC GCC ACA GAA GCA GCC GCC CTT (SEQ IDNO:28) CTT CTT CTT CTT CTT CTT TCC CCC GCA GTC ACA TGA ACC T-3′ Outsideprimer AAV 2/4 5′ top primer: 5′-TGC CGA GCC ATC GAC GTC AGA (SEQ IDNO:29) CGC G-3′

[0260] The RGD linker was inserted into the H2285, R2356, H2591, H2634,H2690, H/N3761, and H/N4047 mutants from Table 1.

[0261] The bradykinin linker was inserted into the H2285, H2416, H2591,H2634, H2690, H/N2944, and H/N3761 mutants from Table 1.

[0262] The poly-Lys linker was inserted into the H2285, H2591, H2690,and H/N3761 mutants from Table 1.

[0263] The poly-His linker was inserted into the H2285, H2416, H2591,H2634, H2690, H/N2944, H3561, H3766, and H/N4047 mutants from Table 1.

EXAMPLE 16 Characterization of Insertion Mutants

[0264] The insertion mutants at site H2690 all have titers similar tothe original 12 bp insert. Using the ELISA assay and the anti-histidineantibody polyHis insertions into this site were shown to be displayed onthe surface of the virion.

[0265] The polyHis epitope was also shown to be on the surface wheninserted into site H2634. Interestingly, the Western blot analysis ofthe 12 bp insertion at H2634 did not show any VP1 or VP2 subunits beingformed. It has been determined that this insertion in VP2 is near thenuclear localization signal for the VP1 And VP2 subunits. It is possiblethat this domain was disrupted by the original insertion, and with theaddition of the 8-histidines the domain was repaired. Although the dotblot of this 8His virus showed the presence of viral particles, theseparticles were not infectious.

[0266] The insertion site H2591 is in VP1. Insertion of linker epitopesinto this site do not affect the titer any more than did the original 12bp insertion at this site (Table 1).

[0267] The insertion at site N4160 is in VP3 near the carboxy terminus.This insertion mutant is of interest because the original 12 bpinsertion infects cell at an equivalent level as wild-type (Table 1).

[0268] Mutant R3317, which as been previously described in Table 1,appeared not to protect virions by dot blot analysis. Repeating thisexperiment with a LacZ transgene, the same results were observed, i.e.,no protected particles. However, when using an independent clone and theGFP transgene (˜1000 bp smaller than LacZ) protected particles wereobserved. In addition, the GFP-expression virion transduced HeLa cellsat high levels, equivalent to wild-type. It is unclear why disparateresults were observed with different transgenes.

[0269] In addition, a linker encoding the respiratory syncitial virusheparin binding domain is inserted into the H2690 mutant at a site thattolerates inserts without loss of viability (Table 1) to restore heparinbinding to this mutant.

EXAMPLE 17 Unique Restriction Site Mutants

[0270] Unique restriction sites within the capsid of AAV type 2 weremade to facilitate the generation of insertional mutants. The sites werechosen so that the mutations introduced into the nucleotide sequence ofthe capsid were conservative, i.e., were not missense mutations orresult in stop codons. Amino acid positions 586, 529, 595, 552, and 517(VP1 methionine as amino acid #1) were chosen. For all of thesepositions, except 529, unique Hpa I sites were engineered. For the siteat amino acid 529, a unique Eco RV site was engineered. Each of theseunique restriction sites results in an in-frame blunt ended digestionproduct. So frame 1 linkers were used to insert into these sites.Overlapping primers were used to generate the unique sites, and outsideprimers were used to generate the right and left fragments of theinsertion.

[0271] The right fragment was then digested with Nsi I and either Eco RVor Hpa I, and the left fragment with Hind III and either Eco RV or HpaI. We cloned these digestion products into the pACG vector that hadalready digested with Hind III and Nsi I. The resulting plasmid was thendigested with Xcm I and Bsi WI. these enzymes result in an ˜750 bpfragment around the engineered unique restriction site. This strategywill result in the accumulation of fewer errors because the PCRgenerated sequences are smaller.

[0272] The primers: 595 top primer 5′-GCA GAT GTT AAC ACA CAA GGC GTT(SEQ ID NO:30) CTT CCA-3′: 595 bottom primer 5′-TTG TGT GTT AAC ATC TGCGGT AGC (SEQ ID NO:31) TGC TTG-3′: 586 top primer 5′-CAG AGA GTT AAC AGACAA GCA GCT (SEQ ID NO:32) ACC GC-3′: 586 bottom primer 5′-GTC TGT TAACTC TCT GGA GGT TGG (SEQ ID NO:33) TAG ATA-3′: Note: This constructresults in a missense mutation Glycine to Valine 552 top primer 5′-ACAAAT GTT AAC ATT GAA AAG GTC (SEQ ID NO:34) ATG ATT-3′: 552 bottom primer5′-TTC AAT GTT AAC ATT TGT TTT CTC (SEQ ID NO:35) TGA GCC-3′: 529 topprimer 5′-GGA CGA TAT CGA AAA GTT TTT TCC (SEQ ID NO:36) TCA G-3′: 529bottom primer 5′-ACT TTT CGA TAT CGT CCT TGT GGC (SEQ ID NO:37) TTGC-3′: Note: This construct results in a missense mutation Glutamic acidto Isoleucine 517 top primer 5′-TCT CTG GTT AAC CCG GGC CCG GGC (SEQ IDNO:38) ATG GCA-3′: 517 bottom primer 5′-GGC CGG GTT AAC CAG AGA GTC TCT(SEQ ID NO:39) GCC ATT-3′: The outside primers were: 5′primer 5′-TGC GCAGCC ATC GAC GTC AGA (SEQ ID NO:40) CGC G-3′: 3′primer 5′-CAT GAT GCA TCAAAG TTC AAC TGA (SEQ ID NO:41) AAC GAA T-3′:

[0273] Four clones were also generated with the RGD and 8His linkers(Example 15) inserted into the 529 Eco RV site. Five 8His linkers andone RGD linker insertion mutants were generated into the 586 Hpa I site.

[0274] The unique restriction site messense mutations at 3790-3792(amino acid 529; EcoRV) did infect HeLa cells, although at relativelylow efficiency (˜{fraction (1/100)} to˜{fraction (1/1000)} ofwild-type). When the 8His epitome insert was inserted at this site, theresulting virus had a lip phenotype (i.e., a low infectious particle).

[0275] Insertions into the unique missense restriction site at 3690-3961(amino acid 586; Hpa I) both 8His and RGD were both very infectious,transducing HeLa cells at least as well as wild-type virus.

EXAMPLE 18 Double Mutants

[0276] Double mutants were generated using the single mutant H3761(Table 1) as a template. The H3761 insertion mutant does not bindheparin sulfate as assessed by both batch and column bindingexperiments. This mutant is interesting because it does not infect anyof the cell lines so far tested, although electron microscopy analysissuggests that this virus forms normal parvovirus shells, and by dot blothybridization this virus packages the viral genome efficiently.

[0277] The region of the capsid coding the sequence that contains theH3761 insertion was subcloned into other insertion mutants to createdouble-mutants. The H2690 (AA# 163) insertion mutant was chosen becauseit has been shown to display a poly-His insertion epitopes on the viralsurface (as assessed by using the conformational specific antibody tobind the virus to an ELISA plate and an anti-histidine antibodypreconjugated to horse radish peroxidase to detect the virus containinghistidines).

[0278] the H2690 insertion mutant helper plasmid (pACG H2690 BRDY)containing the bradykinin insertion (Example 15) and the pACG H3761insertion mutant were both digested with Hind III and Bsi WI. The HindIII site is in the rep gene, while the Bsi WI site is between 2690 and3761. The small fragment contains pACG H2690 BRDY while the largefragment contains pACG H3761.

[0279] A double mutant H2690 BRDY H3761, with the bradykinin insertinserted at the H2690 site, demonstrated a five-fold increase ininfectivity of A9 cells expressing the bradykinin receptor as comparedwith the parental A9 cells alone. These results indicate (1) the defectin binding of the H3761 is likely at the point of binding to cellular HSreceptors, but this virus retains infectivity if directed into cells byanother route, and (2) the bradykinin double-mutant targeted entry ofthe virus into bradykinin-receptor expressing cells.

[0280] The H3761 insertion mutant has also been cloned into the uniquerestriction site missense mutations (Example 17), AA# 586 (Hpa I) andAA# 529 (EcoRV). The restriction enzyme NcoI lies between the H3761 andthe 529 (glu→Ile) and 586 (Gly→Val) missense mutations, and this enzymecuts within the rep gene. By digesting the pACG2 helper plasmid containthe H3761 and the 586 and 529 unique sites with Nco I, the small Nco Ifragment (3142 bps) containing the H3761 insertion mutation and thelarge Nco I fragment (5034 bps) containing the 586 and 529 unique siteswere isolated. After ligation, the constructs with the correctorientation were established, and these clones were used to make virus.

[0281] the unique restriction site missense mutations that containingthe RGD motif (Example 15) were also used in this cloning strategy.Thus, there are double mutants containing no inserts at the unique sitesand double mutants containing RGD epitopes at those sites.

[0282] The H3761 mutant does not transduce HeLa or CHO-K1 cells. Incontrast, the 586-RGD double mutants exhibited transduction of both ofthese cell types. These results strongly suggest that the transductionwas mediated by the RGD motif introduced into the 586 unique restrictionsite.

[0283] The double mutants with the unique restriction sites, but noinserts, and the 529-RGD double mutant did not exhibit efficienttransduction of HeLa or CHO-K1 cells.

EXAMPLE 19 MSH-Targeted AAV Vector

[0284] In one embodiment of the invention, melanocyte stimulatinghormone (MSH) is used for targeting of AAV vectors to cells expressingMSH receptors. Studies have shown that this peptide will directligand-associated complexes specifically into melanocyte NEL-M1 cells(Murphy et al., (1986) Proc. Nat. Acad. Sci USA 83:8258), providing aconvenient test system. For example, diphtheria toxin tethered to a12-residue peptide encoding the MSH ligand was efficient in killing onlyMSH receptor expressing cells (Morandini et al., (1994) Internat. J. Ca.56: 129) Cell death was attributed to receptor mediated endocylosis ofthe specific ligand delivery.

[0285] MSH is inserted into loop 3 of the AAV type 2 capsid. In thefirst step, an AAV type 2 deletion mutant is made with a 12-amino aciddeletion when the Bgl II—SpH I fragment is removed from the sequenceencoding loop 3. The sequence encoding the MSH peptide is then insertedinto the deleted region.

[0286] The primer sequences to make the loop 3 and loop4 insertionmutations are as follows:

[0287] Loop 3 5′ top primer (SEQ IDNO:42):5-′GATACCTTAAGATCTAGTGGAACCACCACGCACTCAAGGCTT-3′25

[0288] The cttaag is an Afl II site, the agatct is a Bgl II site. Thesetwo sites overlap by two base pairs. The homology with the AAV sequencestarts at position 3556 and ends at 3583.

[0289] Loop 3 3′ bottom primer (SEQ IDNO:43):5′CTAGCTTAAGCATGCATACAGGTACTGGTCGATGAGAGGATT-3′

[0290] The gcatgc is a Sphl site, and the cttaag is an Afl II site.These two sites overlap by one base pair. The homology with the AAVsequence starts at position 3505 and ends at 3531 (note that this is thebottom strand).

[0291] These primers remove 24 bp (i.e., 8 amino acids) of AAV type 2sequences from 3532 to 3555. The deleted amino acid sequence is Tyr LeuSer Arg Thr Asn Thr Pro from at amino acid 444 to 451 (VP1-Met beingamino acid #1).

[0292] the 5′Sph I Afl II Bgl II 3′ sites in the sequence:5′-GCATGCTTAAGATCT-3′ result in the addition of 5 amino acids Ala CysLeu Arg Ser.

[0293] Virus is produced by standard packaging methods. The MSH-taggedAAV type 2 vector is evaluated for transduction in HeLa cells and cellswith MSH receptors (e.g., melanocytes).

EXAMPLE 20 Chimeric AAV2/4 Virus—Capsid Protein Substitutions

[0294] The virions of the AAV serotypes are made up of three proteinsubunits VP1 VP2 and VP3. VP3 is the most abundant subunit, itrepresents between 80-90% of the 60 subunits that make up the virion,with VP1 and VP2 making up 5-10% each of the virion. The subunits aretranslated from an overlapping transcript, so that VP3 sequences arewithin both VP2 and VP1, and VP2 sequences are within VP1.

[0295] We have designed primers that enabled us to substitute entiresubunits and unique domains of subunits between AAV2 and AAV4. AAV4 hasproperties that are significantly different from AAV2. Thus, definingthe domains that account for these distinct properties would be ofvalue, e.g., for designing gene therapy vectors.

[0296] We have chosen a seamless cloning strategy to clone the subunitsor unique domains of subunits between these two serotypes. AAV2 and AAV4top primer 5′-TGC CGA GCC ATC GAC GTC AGA CGC (SEQ ID NO:44) G-3′: AAV2and AAV4 bottom primer 5′-CAT GAT GCA TCA AAG TTC AAC TGA (SEQ ID NO:45)AAC GAA T-3′: AAV2 VP3 top primer 5′-CGA GCT CTT CGA TGG CTA GAG GCA(SEQ ID NO:46) GTG GCG GAC-3′: AAV2 VP3 bottom primer 5′-AGC GCT CTT CCCATC GTA TTA GTT (SEQ ID NO:47) CCC AGA CCA GAG-3′: AAV2 VP2 top primer5′-CGA GCT CTT CGA CGG CTC CGG GAA (SEQ ID NO:48) AAA AGA GGC-3′: AAV2VP2 bottom primer 5′-AGC GCT CTT CCC GTC TTA ACA GGT (SEQ ID NO:49) TCCTCA ACC AGG-3′: AAV4 VP3 top primer 5′-CGA GCT CTT CGA TGC GTG CAG CAG(SEQ ID NO:50) CTG GAG GAG CTG-3′: AAV4 VP3 bottom primer 5′-AGC GCT CTTCGC ATC TCA CTG TCA (SEQ ID NO:51) TCA GAC GAG TCG-3′: AAV4 VP2 topprimer 5′-CGA GCT CTT CGA CGG CTC CTG GAA (SEQ ID NO:52) AGA AGA GAC-3′:AAV4 VP2 bottom primer 5′-AGC GCT CTT CCC GTC TCA CCC GCT (SEQ ID NO:53)TGC TCA ACC AGA- 3′:

[0297] These primers will result in the subunit swaps that are shown inFIG. 7. A representative sequence of a chimeric AAV2 capsid in which theAAV4 Vp2 was substituted is shown in Appendix 2 (SEQ ID NO:2). Thissequence contains the AAV2 rep coding sequences, most of the AAV2 Vp1and Vp3 coding sequences, and the entire AAV4 Vp2 coding sequences andsome of the AAV4 Vp1 and Vp3 coding sequences in a pBluescript backbone.

[0298] The Rep68/78 coding sequence begins at nu 251 of SEQ ID NO:2, andthe Rep52/40 coding sequence begins at nu 923. The Rep78/52 stop signalends at nu 2114, and the stop for Rep68/40 is at nu 2180. The capsidcoding sequence starts at nu 2133 and the end at nu 4315 (Vp1 start atnu 2133, Vp2 start at nu 2544, Vp3 start at 2724).

[0299] The AAV2 sequences from the second XhoI site at bp 2420 in Vp1tothe Bsi WI site at bp 3255 in Vp3 in the AAV2 cap genes was replacedwith the corresponding region from AAV4 (corresponding to nu 2350-3149in the plasmid sequence). Briefly, the AAV2 helper plasmid pACG2 waspartially digested with XhoI and Bsi WI releasing the 835 bp fragment.The same digest in AAV4 resulted in a 799 bp fragment that was ligatedinto the deleted AAV2 sequence to produce the helper virus encoding thechimeric AAV2/4 capsid.

[0300] Virions are produced carrying a recombinant AAV genome,preferably a recombinant AAV2 genome, typically expressing a reportergene (e.g., GFP). These mutant viral vectors are characterized forvirion formation, morphology, genome protection, heparin binding, andinfectivity as described in Example 15.

EXAMPLE 21 Construction of B19/AAV-2 Chimeric Vectors

[0301] Studies by Dong et al., (1996) Human Gene Therapy 7:2101, havedetermined the packaging limitations using rAAV vectors. Usingrecombinant AAV DNA templates with increasing insertions of stuffer DNA,Dong et al. determined that the packaging capacity of rAAV vectorsdeclined dramatically between 104% and 108% of wt (4883 vs. 5083nucleotides, respectively). This packaging restriction precludes the useof important genes, including mini muscular dystrophy genes as well aspromoter regulated cystic fibrosis sequences.

[0302] Accordingly, the present investigations set out to develop aB19/AAV-2 derived gene therapy vector that maintains the packagingcapacity of B19, the tropism of AAV-2, as well as function as asubstrate for targeting vectors. The human parvovirus B19 (packagingcapacity of 5.6 kb) was chosen to utilize the major structural proteinVp2 in the generation of a chimeric AAV vector for packaging largervector genomes. B19 is composed of only two overlapping structuralproteins (Vp1 & 2). B19 infects primary erythroid progenitor cells usinggloboside as its receptor (Brown et al., (1993) Science 262:114). Thestructure of B19 has been determined to 8Δ Aresolution (Agbandje-McKennaet al., (1994) Virology 203:106).

[0303] A chimeric AAV particle was constructed by swapping the AAV majorstructural protein Vp3 for B19's Vp2. Seamless cloning (Stratagene USA)was utilized to generate an AAV helper construct that would express allof the AAV proteins (Rep 78, 68, 52, 40 and Vp 1 and Vp2) with B19substituted for the Vp3 major Cap protein (FIG. 8; nucleotide sequencein Appendix 3 and SEQ ID NO:3; amino acid sequence in Appendix 4 and SEQID NO:4).

[0304] The starting material for the chimeric vector was pAAV-Ad andpYT103c. pYT103c contains the entire B19 coding domain without terminalrepeats. HindIII digestion of pAAV-Ad released a 2727bp fragment whichcontained the entire AAV2 capsid coding region and some flankingregions. This fragment was subcloned into Hind III digestedpBS+(Stratagene), resulting in pBS+AAVCap. Polymerase chain reaction wasused to amplify the Vp2 coding region from pYT1 03c. The primers were5′-AGTTACTCTTCCATGACTTCAGTTAATTCTGCAGAA 3′(SEQ ID NO:54) in the5′direction and 5′-AGTTACTCTTCTTTACMTGGGTGCACACGGCTTTT 3′(SEQ ID NO:55)in the 3′direction. Primers to pBS+AAVCap were used to amplify aroundVp3 of AAV2. The primers were 5′AGTTACTCTTCTTMTCGTGGACTTACCGTGGATAC 3′(SEQ ID NO:56) in the 5′direction and5′-AGTTACTCTTCCCATCGTATTAGTTCCCAGACCAGA 3 (SEQ ID NO:57), in the3′direction. Six nucleotides from the 5′end of each primer is an Eam1104 I site, this site digests downstream from its recognition site inthis case the overlap is an ATG and its compliment and a TAA and itscompliment. This site is utilized during the seamless cloning strategy(Stratagene). Digestion of B19-Vp2 and AAV2 PCR products with Eam 1104-Iand cloning resulted in a subclone of pBS+AAVCap with Vp2 of B19substituted for AAV2 Vp3. This vector was digested with Hind III andcloned back into pAAV-Ad and orientation determined resulting inpAAV/B19-Ad (Appendix 3; SEQ ID NO:3). This sequence encodes region(start at nt 1), followed by the AAV2 Vp2 region (start at nt 412), andthen the B19 Vp2 region (start at nt 607).

EXAMPLE 22 Production of Chimeric Virus

[0305] The pAAV/B19 helper construct was used in a transient packagingsystem as described in Example 1. Briefly, the helper plasmidspAAV/B19-Ad and pAB11 (which contains AAV2 terminal repeats and theβ-galactosidase gene under the control of the CMV early promoter) wereco-transfected into 293 cells by calcium phosphate transfection themedium was changed and adenovirus dl309 (MOI-5) was added. Forty-eighthours later the cells were centrifuged and the supernatant wasdiscarded. A fraction of the cell pellet was used in a HIRT assay. Thecell pellet was lysed in cesium chloride (1.39 g/ml), sonicated andcentrifuged at 41,000 rpm for 72 hours. Fractions from the cesiumgradient were recovered and samples from each were used in dot blothybridization to test particle number of virus. The dot blots wereprobed with β-galactosidase gene, and particle numbers were determinedby control amounts of the β-galactosidase gene. Peak fractionscontaining virus were dialysed against PBS, 20% glycerol.

EXAMPLE 23 Infection of Cells with Chimeric Virus

[0306] Forty-eight hours post-transfection, cell lysates were generatedand tested for transduction into various target cells. A transducingtiter of 2×10⁶ was generated. Various volumes of virus were added to293, RT-2 rat glioma, U-87 glioma, as well as to two primary humanglioblastoma cell lines in small volumes of medium. Virus was also addedto UT7 megakaryoctye cells that had been incubated in the presence oferythropoeitin (EPO) for several weeks. Exposure of UT7 cells to EPO isknown to render these cells permissive for B19 infection.

[0307] Adenovirus was also added to the cells at an MOI of 5. Two hoursafter infection the virus was washed off and fresh medium was added.Twenty-four hours post infection the cells were washed with PBS, fixedin formaldehyde/gluteraldehyde, and stained with X-gal. Twelve totwenty-four hours later the number of blue cells was determined bycounting ten fields.

[0308] Transduction was obtained in the glioma and primary humanglioblastoma cells. Efficient transduction was not observed in 293 cells(a cell type typically infected with AAV). Interestingly, transductionwas seen with the UT7 cells. These results suggest that the chimera haslost the native AAV tropism and has acquired the B19 tropism forerythroid cells. This virus is characterized to determine whether it hasretained the antigenic properties associated with the AAV2 serotype.

[0309] The B19 globoside binding region (loop 4 between amino acids399-406 of the Vp2 subunit; Brown et al. (1993 Science 262:114 virus ofthis is chimeric virus is deleted, modified or swapped out to reduce orcompletely eliminate the B19 tropism for erythroid cells.

EXAMPLE 24 Characterization of B19/AAV Chimera

[0310] The results from Example 23 indicate that a transducing chimericvirus was successfully generated. The chimeric virus was furtherevaluated for total particle yield and integrity. The remainder of thevector preparation was gradient purified, and the chimeric virus wasanalyzed by dot blot analysis to determine a particle titer of 1×10⁸ andEM analysis (see Example 6) to determine if a correct icosahedralstructure was formed (FIG. 9). From this analysis, it was confirmed thatthe chimeric virion that was generated retained the typical parvovirusstructure and was stable to physical purification step such assonication and CsCl₂ gradient centrifugation. This is an importantobservation since most parvovirus are heat stable (resistant up to 65degrees), resistant to detergents (0.5% SDS) and can tolerate extreme pHchanges (viable between pH 2.0-11).

[0311] In addition, EM analysis yielded unexpected results (FIG. 9).Virion particles of two different sizes were observed (a 23-28 nmparticle, typical for wt AAV, and a 33-38 nm particle, never beforeidentified). Further analysis suggested that the AAV 33-38 nm particlewas formed by changing the triangulation number from T=1 to T=3,resulting in larger particles containing 180 copies of the major capsidcomponent instead of 60. These surprising results indicate that a virionstructure larger than wt AAV has been generated. This virion may havethe potential for carrying larger than wt vector templates. The larger33-38 nm particle will be useful in increasing packaging limits abovethe 6 kb range (the B19 25 nm particle packages 6 kb of DNA).

EXAMPLE 25 Packaging Capacity of B19/AAV-2 Chimera

[0312] To quantitate the packaging capacity of the chimeric virus fromExample 21, a series of vectors developed by Dong and coworkers, (1996)Human Gene Therapy 7:2101, is utilized with genomes of progressivelyincreased sizes having inserts between 745 and 1811 bases (for a maximumtotal genome size of 6.4 kb). Small-scale production of chimericrecombinant virus is used to assay packaging efficiency by testing theDNA content of the virus using Hirt assay, and by chloramphenicolacetyltransferase (CAT) reporter assay.

EXAMPLE 26 Construction of Other B19/AAV Chimeras

[0313] Other chimeric B19/AAV capsids are generated as in Example 21(e.g., swapping AAV Vp1 or Vp2 with B19 Vp1) and are characterized asdescribed in Examples 22-25 above. In particular, both B19 Vp1 and Vp2are substituted into an AAV Vp1 chimera to generate a novel chimericcapsid containing AAV Vp1 and B19 Vp1 and Vp2.

[0314] These chimeras are assayed in 293 (typically infected by AAV) anderythroid cells (the cell type typically infected by B19) fortransduction efficiency and are assayed for packaging recombinant AAVvectors with increasing sized inserts as described above.

[0315] If desired, the B19 globoside binding region (loop 4 of Vp2between amino acids 399-406; Brown et al. (1993) Science 262:114) ofthese vectors can be deleted, modified or swapped out to remove the B19tropism.

EXAMPLE 27 Loop Swaps Between AAV Serotypes

[0316] The capsid gene of AAV2, in the helper vector pACG2, was digestedwith the enzymes Asp718 and Bsi WI. Bsi WI has a unique site in the AAV2genome at position 3254 bp, and Asp718 digests the genome twice at 1906and 4158 bps (AAV2 sequence numbers). The capsid coding domain of AAV2was partially digested with Asp718 and the full length (single cut)fragment was isolated. This fragment was then digested with Bsi WI andthe 7272 bp fragment isolated. This fragment removed the 904 bp fragmentthe contains the coding region of the VP3 loop 2, 3, and 4 domains.

[0317] The capsid gene of AAV4 was digested with Asp718 and Bsi WI tocompletion and a 928 bp fragment from 3284 bp (BsiWI) to 4212 bps(Asp718) was isolated (AAV4 sequence numbers). This AAV4 fragment codesfor a region in VP3 that contains loops 2, 3 and 4. The 928 bp AAV4fragment and the 7272 bp fragment from pACG2 were ligated and cloneswere identified.

[0318] These clones were used to make a chimeric virus that containedmostly AAV2 and part of the VP3 domain of AAV4. This virus did notinfect HeLa cells as determined by blue stained cells (viral infectedcells expressing the LacZ marker gene). However, like AAV4 these cellsinfected COS7 cells at a low titer of 1×10⁵ transducing units/mL. Thesevirions are not recognized by the AAV2 monoclonal antibody B1

[0319] Chimeric virus was also made in which Vp3 Loops 2-4 from AAV2were substituted into the homologous region of the AAV4 capsid.

[0320] The AAV3 capsid coding region containing the VP3 loops 2-4domains were cloned into pACG2 in the same manner as described above forAAV2/4 loop swaps. These chimeric AAV2/3 virions bind heparin agaroseand infect HeLa and 293 cells. Furthermore, these virions are recognizedby the B1 monoclonal antibody.

[0321] Likewise, using the techniques taught above, Vp3 loops 2-4 fromAAV5 are substituted for loops 2-4 of AAV2.

[0322] Furthermore, single loops (e.g., loop 2, 3 or 4, or loops 2-3 or3-4) are substituted from AAV3, 4 or 5 into AAV2 or vice versa.

[0323] These mutant viral vectors are characterized for virionformation, morphology, genome protection, heparin binding, andinfectivity as described in Example 4-7.

[0324] A representative helper plasmid encoding a chimeric AAV2/3 capsidis given in Appendix 5 (SEQ ID:5). This sequence contains the AAV2 repcoding sequences, most of the AAV2 capsid coding sequences, with theexception that loops 2-4 from the AAV2 Vp3 subunit were replaced withthe corresponding region from AAV3, in a pBluescript backbone. The Rep68/78 coding sequence starts at nu 251, and the Rep52/40 coding sequencestarts at nu 923. The rep coding sequences end at nu 2114 for Rep78/52and at nu 2180 for Rep68/40. The cap coding region starts at nu 2133 andends at nu 4342 (Vp1 start at nu 2133, Vp2 start at nu 2544, Vp3 startat nu 2739).

[0325] Briefly, both AAV2 (pACG2) and AAV3 helper plasmids were digestedwith Bsi WI and Asp 718. This removes a 904 bp fragment in the AAV2genome from nu 3255 to 4159. In the AAV3 genome, the same digestionremoved 907 bp from nu 3261-4168. This 904 bp fragment was ligated intothe deleted AAV2 helper to result in the helper given in SEQ ID NO:5(AAV3 sequences at nu 3184-4092 of the plasmid).

EXAMPLE 28 Hybrid Viruses

[0326] Primers were made to create a unique Hind III site in the AAV4rep gene that overlapped the Hind III site in AAV2. In addition, at the3′end of the rep coding sequence, a unique Not I site was created 3′ ofthe polyadenylation site. A virus purchased from American Type CultureCollection (ATCC) as the template for the PCR.

[0327] The 5′ portion of the AAV2 rep gene from the Xba I site to theHind III site was subcloned into pBluescript. The Hind III-Not I PCRdigestion product was then cloned into the pBluescript containing the 5′rep gene digested with Not I and Hind III. Primers: AAV4 3′Not I primer5′-AAG CGC CGC GGC CGC TGC TTA TGT (SEQ ID NO:58): ACG CA-3′ AAV4 5′HindIII primer 5′-GAC GCG GAA GCT TCG GTG GAC TAC (SEQ ID NO:59): GCG-3′

[0328] This cloning strategy resulted in a helper plasmid that is ahybrid for AAV2 and AAV4 rep genes and contains the AAV4 cap genes. Thishelper contains the AAV2 rep gene up to the Hind III site and from thispast the polyadenylation site the sequences are derived from AAV4.

[0329] This virus packaged a recombinant AAV2 genome with AAV2 ITRs.This hybrid AAV2/4 virus exhibits the binding characteristics of AAV4,e.g., it does not bind HS and transduces AAV4 target cells that are nottypically permissive to AAV2 transduction.

[0330] The hybrid AAV 2/4 helper plasmid is as given in Appendix 1(SEQID NO:1). This sequence encodes the AAV2 rep genes and AAV4 capsid in apBluescript backbone. The Rep 68/78 coding sequence starts at nu 251,and the Rep52/40 coding sequence starts at nu 923. The rep codingsequences end at nu 2120 for Rep78/52 and at nu 2183 for Rep68/40. Thecap coding region starts at nu 2123 and ends at nu 4341 (Vp1 start at nu2123, Vp2 start at nu 2547, Vp3 start at nu 2727).

[0331] Using the same techniques, a hybrid AAV2/3 virus in which arecombinant AAV2 genome (with AAV2 ITRs) is packaged. The resultinghybrid virus is viable and efficiently transduces AAV3 permissive cells.

[0332] In addition, in contrast to a recent report (Chiorini et al.,1999) J. Virology 73:1309), the techniques described above have beenused to produce a hybrid AAV2/5 virus in which a recombinant AAV2 genome(with AAV2 ITRs) is packaged within a AAV Type 5 capsid. This virus ispackaged relatively inefficiently, but the resulting particlesdemonstrated transduction of cells.

[0333] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claimsand equivalents thereof. APPENDIX 1CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCGCTGCTTATGTACGCAGTAGCCATGGAAACGAGATAAGATAAGAAGGACACGGAGACCAAAGTTCAACTGAAACGAATAAACCGGTTTATTGATTAACAGGTTATTACAGGTGGTGGGTGAGGTAGCGGGTACCGATAGCCCTAGGCTCAGTGTATTTCCCAGCCGCATCGGGAGCCCACAACAGAGAGTTTTGCTGTCCGTAGTTGGAGGTAAACTGGACCTCGGGGTTCCAGCGTTTGGACCGCTCCTTCTGGATCTCCCAGTCAATCTGCACCGACACCTGGCCAGTGCTGTACTGAGTAATGAAGGAGTTTACCGGAGTAGAGCTGAAGGTCGTTGCAGGATTCGCAGGTACCGGGGTGTTCTTGATAAAAATTTGAGGAGGCGGGTGTTTCAGCCCAAACCCACCAATCAGCGGTGAGGGGTGAAAGTGTCCATCGGTATGAGGAATCTTGGCCCAAATGGGACCCTGGTAGTAAATGTCTCTGTTTTGCCAGACCATTCCAGGCACGGCTCCCAAGGCTGTCAGTCTGTCCACGGTCGGCAGGTTGCTGTTGCTCTGGTCACCGCCAGGTAGGTTGCCCCACATGTCCGTATCGGTGGCGTTGGTGGCTGCCAGCTCCTCCTCAGAGGTGAAGATCAGAGTCCCGGGTACGGTGGCCGTGTTGCCGTTCTGTTTAGGCCCCGCAAAGATGAGCTGGCTGTTGCTGAACTTGCTGTCCGCAGGTCCAGCCGTGGCCATTGGAGGTCCGGGGGTCAGGGCACTCCATCTTCCGTCCAGAGTGCTGTGCGTCTCGTATTTGATGAGACTGTCTGACCCGGTGGCAGGGATCTTGTAGTTTTGATTGGCAGTCTTTGAGAAGCCCTGCTGCTTGATTGAAGGCCCGGGCAGCCAGTTCTTTTTAAAGTTGGAAAAGTTGGTAGGCCGCAGCTTGGTAAAGTTGGTGGTGGCAGTCCCGGCATTCAGGGTGGTTCCGGTGGTGGTCGATTGCAGTCCCCACAGGTACTGGTCGATGAGAGGGTTCATCAGCCGGTCCAGGCTCTGGCTGTGCGCGTACATCGAGTGGAAAGGCACCTTCTCAAAACTGTACGTAATTTCAAAGTTGTTGCCAGTCCGCAGCATCTGCGAAGGAAAGTACTCCAGGCAGTAGAAGGCATTTCTGTCAGTCTGTTGCTGCGAAGTGTTGCCGGTCACCAGTCCACAGTAGCCGTACTGGGGCACCATAAAGACGTCGTTGGGAAAAGGAGGCAGGCTGCCCTCTTGACCCGCATCCATCACGTACGGCAGTTCGTACGACGAGTCCGCAAAGATCTGAACCGTGCTGGTAAGGTTATTAGCCACCGTTGTCTCGCCGTTCGACGTCGTGACCTCCTTGACCTGGATGTTGAAGATTTTGACCCGCATGGCTTTGGGTCGCATGCCCCAGTTGTTGTTGATGAGTCGCTGCCAGTCACGTGGTGAGAAGTGGCAGTGGAAGCGGTTGAAGTCAAAGTATCCCCAGGGGGTGGAGAATCCGTTGTAGGTGTTGGACTGCAGGCTCTCTCCGAGTCGCTTGTAGAGGTGGTTGTTGTAGGTGGGCAAGACCCAGGTTCTGGTGCTGGTGGTCGTGACGTGGCCCTCAGACCAGGTGGAATCGCAATGCCAATCACCCGAGGCATTACCCACTCCATCGGCACCTTGTCCGCCCTCGACTGCAGCTCCGCCAGCTGCTGCACGCATCTCACTGTCATCAGACATGGCTCCGGAAGTTGATCCCTCAGGGGGTCCGTCGCCTGCTCCAGTTTCGTCTTCGAAAACGAGCTTCTTTTTAGCCGGCTGCTTGCCTTTTTTGCCGATACCCGTGGAGGAGTCGGGCTGCTGGGGGGATTCAATCAACGGTCTCTTCTTTCCAGGAGCCGTCTCACCCGCTTGCTCAACCAGACCAAGAGGTTCAAGAACCCTCTTTTTGGCCTGGAAGACTGCTCTGCCGAGGTTGCCCCCAAACGATGTGTCGCCCTGAAGCCGCTGCTGGAACTCCGCGTCGGCGTGGTTGTACTTGAGGTAGGGGTTGTCACCGGCCTTGAGCTGCTGGTCGTAGGCCTTGTCGTGCTCGAGGGCTGCCGCGTCCGCTGCGTTGACGGGTTCCCCCTTGTCGAGTCCGTTGCCGGGTCCGAGGTATTTGTAACCCGGAAGCACAAGACCCCGAGCGTTGTCCTGATGTTGTTGATTTGCCTTGGGTTTAGGGGCTCCAGGTTGCAGCGCCCACCACTCTCGAACGCCTTCAGAGAGGTTGTCCTCTAGCCAATCTGGAAGGTAACCGTCAGTCATATCTGGTTTGAGTCATTTATTGTTCCATGTCACAGTCATCCAAGTCCACATTGGCCAGTTCGCAGGCCGAGCAGGCCACCTCGGGCGCCCTCCCCATGATGTGATGAATCGGACACAGTTTCTGATACGTCCGCTTTCTGACGACAGACACGGGTTGAGATTCTGACACGGGGAAGCACTCGGCACAGTCCATGACCCCGTGCGTGAAGCAAATGTCCACATTCTGATTCATTCTCTCGCATTGCCGGCAGGGAAAAAGCATCAGATTCATACCCACGTGACGAGAACATTTGTTTTGGTACCTGTCCGCGTAGTCCACCGAAGCTTCCGCGTCTGACGTCGATGGCTGCGCAACTGACTCGCGCACCCGTTTGGGCTCACTTATATCTGCGTCACTGGGGGCGGGTCTTTTCTTGGCTCCACCCTTTTTGACGTAGAATTCATGCTCCACCTCAACCACGTGATCCTTTGCCCACCGGAAAAAGTCTTTGACTTCCTGCTTGGTGACCTTCCCAAAGTCATGATCCAGACGGCGGGTGAGTTCAAATTTGAACATCCGGTCTTGCAACGGCTGCTGGTGTTCGAAGGTCGTTGAGTTCCCGTCAATCACGGCGCACATGTTGGTGTTGGAGGTGACGATCACGGGAGTCGGGTCTATCTGGGCCGAGGACTTGCATTTCTGGTCCACGCGCACCTTGCTTCCTCCGAGAATGGCTTTGGCCGACTCCACGACCTTGGCGGTCATCTTCCCCTCCTCCCACCAGATCACCATCTTGTCGACACAGTCGTTGAAGGGAAAGTTCTCATTGGTCCAGTTTACGCACCCGTAGAAGGGCACAGTGTGGGCTATGGCCTCCGCGATGTTGGTCTTCCCGGTAGTTGCAGGCCCAAACAGCCAGATGGTGTTCCTCTTGCCGAACTTTTTCGTGGCCCATCCCAGAAAGACGGAAGCCGCATATTGGGGATCGTACCCGTTTAGTTCCAAAATTTTATAAATCCGATTGCTGGAAATGTCCTCCACGGGCTGCTGGCCCACCAGGTAGTCGGGGGCGGTTTTAGTCAGGCTCATAATCTTTCCCGCATTGTCCAAGGCAGCCTTGATTTGGGACCGCGAGTTGGAGGCCGCATTGAAGGAGATGTATGAGGCCTGGTCCTCCTGGATCCACTGTTCTCCGAGGTAATCCCCTTGTCCACGAGCCACCCGACCAGCTCCATGTACCTGGCTGAAGTTTTTGATCTGATCACCGGCGCATCAGAATGGGATTCTGATTCTCTTTGTTCTGCTCCTGCGTCTGCGACACGTGCGTCAGATGCTGCGCCACCAACCGTTTACGCTCCGTGAGATTCAAACAGGCGCTTAAATACTGTTCCATATTAGTCCACGCCCACTGGAGCTCAGGCTGGGTTTTGGGGAGCAAGTAATTGGGGATGTAGCACTCATCCACCACCTTGTTCCCGCCTCCGGCGCCATTTCTGGTCTTTGTGACCGCGAACCAGTTTGGCAAAGTCGGCTCGATCCCGCGGTAAATTCTCTGAATCAGTTTTTCGCGAATCTGACTCAGGAAACGTCCCAAAACCATGGATTTCACCCCGGTGGTTTCCACGAGCACGTGCATGTGGAAGTAGCTCTCTCCCTTCTCAAATTGCACAAAGAAAAGGGCCTCCGGGGCCTTACTCACACGGCGCCATTCCGTCAGAAAGTCGCGCTGCAGCTTCTCGGCCACGGTCAGGGGTGCCTGCTCAATCAGATTCAGATCCATGTCAGAATCTGGCGGCAACTCCCATTCCTTCTCGGCCACCCAGTTCACAAAGCTGTCAGAAATGCCGGGCAGATGCCCGTCAAGGTCGCTGGGGACCTTAATCACAATCTCGTAAAACCCCGGCATGGCGGCTGCGCGTTCAAACCTCCCGCTTCAAAATGGAGACCCTGCGTGCTCACTCGGGCTTAAATACCCAGCGTGACCACATGGTGTCGCAAAATGTCGCAAAACACTCACGTGACCTCTAATACAGGACTCTAGCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGCCAC

[0334] APPENDIX 2 AATTCCCATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTCTCTAGAGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGGGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAAGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGTGACAACCCCTACCTCAAGTACAACCACGCCGACGCGGAGTTCCAGCAGCGGCTTCAGGGCGACACATCGTTTGGGGGCAACCTCGGCAGAGCAGTCTTCCAGGCCAAAAAGAGGGTTCTTGAACCTCTTGGTCTGGTTGAGCAAGCGGGTGAGACGGCTCCTGGAAAGAAGAGACCGTTGATTGAATCCCCCCAGCAGCCCGACTCCTCCACGGGTATCGGCAAAAAAGGCAAGCAGCCGGCTAAAAAGAAGCTCGTTTTCGAAGACGAAACTGGAGCAGGCGACGGACCCCCTGAGGGATCAACTTCCGGAGCCATGTCTGATGACAGTGAGATGCGTGCAGCAGCTGGCGGAGCTGCAGTCGAGGGCGGACAAGGTGCCGATGGAGTGGGTAATGCCTCGGGTGATTGGCATTGCGATTCCACCTGGTCTGAGGGCCACGTCACGACCACCAGCACCAGAACCTGGGTCTTGCCCACCTACAACAACCACCTCTACAAGCGACTCGGAGAGAGCCTGCAGTCCAACACCTACAACGGATTCTCCACCCCCTGGGGATACTTTGACTTCAACCGCTTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGCATGCGACCCAAAGCCATGCGGGTCAAAATCTTCAACATCCAGGTCAAGGAGGTCACGACGTCGAACGGCGAGACAACGGTGGCTAATAACCTTACCAGCACGGTTCAGATCTTTGCGGACTCGTCGTACGAACTGCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGAACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCGGAACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATCGTGGACTTACCGTGGATACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGACTACTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATGCATCGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGAATTCCAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATATTTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATTTTCTCACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTGGAATTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG TTGGCCGATTCATTAATGCA

[0335] APPENDIX 3 ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGACTTCAGTTAATTCTGCAGAAGCCAGCACTGGTGCAGGAGGGGGGGGCAGTAATTCTGTCAAAAGCATGTGGAGTGAGGGGGCCACTTTTAGTGCTAACTCTGTAACTTGTACATTTTCCAGACAGTTTTTAATTCCATATGACCCAGAGCACCATTATAAGGTGTTTTCTCCCGCAGCGAGTAGCTGCCACAATGCCAGTGGAAGGAGGCAAAGGTTTGCACCATCAGTCCCATAATGGGATACTCAACCCCATGGAGATATTTAGATTTTAATGCTTTAAATTTATTTTTTTCACCTTTAGAGTTTCAGCACTTAATTGAAAATTATGGAAGTATAGCTCCTGATGCTTTAACTGTAACCATATCAGAAATTGCTGTTAAGGATGTTACAGACAAAACTGGAGGGGGGGTACAGGTTACTGACAGCACTACAGGGCGCCTATGCATGTTAGTAGACCATGAATACAAGTACCCATATGTGTTAGGGCAAGGTCAGGATACTTTAGCCCCAGAACTTCCTATTTGGGTATACTTTCCCCCTCAATATGCTTACTTAACAGTAGGAGATGTTAACACACAAGGAATTTCTGGAGACAGCAAAAAATTAGCAAGTGAAGAATCAGCATTTTATGTTTTGGAACACAGTTCTTTTCAGCTTTTAGGTACAGGAGGTACAGCAACTATGTCTTATAAGTTTCCTCCAGTGCCCCCAGAAAATTTAGAGGGCTGCAGTCAACACTTTTATGAAATGTACAATCCCTTATACGGATCCCGCTTAGGGGTTCCTGACACATTAGGAGGTGACCCAAAATTTAGATCTTTAACACATGAAGACCATGCAATTCAGCCCCAAAACTTCATGCCAGGGCCACTAGTAAACTCAGTGTCTACAAAGGAGGGAGACAGCTCTAATACTGGAGCTGGAAAAGCCTTAACAGGCCTTAGCACAGGTACCTCTCAAAACACTAGAATATCCTTACGCCCTGGGCCAGTGTCTCAGCCATACCACCACTGGGACACAGATAAATATGTCACAGGAATAAATGCCATTTCTCATGGTCAGACCACTTATGGTAACGCTGAAGACAAAGAGTATCAGCAAGGAGTGGGTAGATTTCCAAATGAAAAAGAACAGCTAAAACAGTTACAGGGTTTAAACATGCACACCTACTTTCCCAATAAAGGAACCCAGCAATATACAGATCAAATTGAGCGCCCCCTAATGGTGGGTTCTGTATGGAACAGAAGAGCCCTTCACTATGAAAGCCAGCTGTGGAGTAAAATTCCAAATTTAGATGACAGTTTTAAAACTCAGTTTGCAGCCTTAGGAGGATGGGGTTTGCATCAGCCACCTCCTCAAATATTTTTAAAAATATTACCACAAAGTGGGCCAATTGGAGGTATTAAATCAATGGGAATTACTACCTTAGTTCAGTATGCCGTGGGAATTATGACAGTAACTATGACATTTAAATTGGGGCCCCGTAAAGCTACGGGACGGTGGAATCCTCAACCTGGAGTATATCCCCCGCACGCAGCAGGTCATTTACCATATGTACTATATGACCCCACAGCTACAGATGCAAAACAACACCACAGACATGGATATGAAAAGCCTGAAGAATTGTGGACAGCCAAAA GCCGTGTGCACCCATTGTAA

[0336] APPENDIX 4 M A A D G Y L P D W L E D T L S E G I R Q W W K L K PG P P P P K P A E R H K D D S R G L V L P G Y K Y L G P F N G L D K G EP V N E A D A A A L E H D K A Y D R Q L D S G D N P Y L K Y N H A D A EF Q E R L K E D T S F G G N L G R A V F Q A K K R V L E P L G L V E E PV K T A P G K K R P V E H S P V E P D S S S G T G K A G Q Q P A R K R LN F G Q T G D A D S V P D P Q P L G Q P P A A P S G L G T N T M T S V NS A E A S T G A G G G G S N S V K S M W S E G A T F S A N S V T C T F SR Q F L I P Y D P E H H Y K V F S P A A S S C H N A S G K E A K V C T IS P I M G Y S T P W R Y L D F N A L N L F F S P L E F Q H L I E N Y G SI A P D A L T V T I S E I A V K D V T D K T G G G V Q V T D S T T G R LC M L V D H E Y K Y P Y V L G Q G Q D T L A P E L P I W V Y F P P Q Y AY L T V G D V N T Q G I S G D S K K L A S E E S A F Y V L E H S S F Q LL G T G G T A T M S Y K F P P V P P E N L E G C S Q H F Y E M Y N P L YG S R L G V P D T L G G D P K F R S L T H E D H A I Q P Q N F M P G P LV N S V S T K E G D S S N T G A G K A L T G L S T G T S Q N T R I S L RP G P V S Q P Y H H W D T D K Y V T G I N A I S H G Q T T Y G N A E D KE Y Q Q G V G R F P N E K E Q L K Q L Q G L N M H T Y F P N K G T Q Q YT D Q I E R P L M V G S V W N R R A L H Y E S Q L W S K I P N L D D S FK T Q F A A L G G W G L H Q P P P Q I F L K I L P Q S G P I G G I K S MG I T T L V Q Y A V G I M T V T M T F K L G P R K A T G R W N P Q P G VY P P H A A G H L P Y V L Y D P T A T D A K Q H H R H G Y E K P E E L WT A K S R V H P L *

[0337] APPENDIX 5 AATTCCCATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTCTCTAGAGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGGGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCCGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTGCTCGGGTCGGCGCACCAAGGCTGTCTCCCGCCGTTTCCAGCGGACGTCTTCATGGTCCCTCAGTATGGATACCTCACCCTGAACAACGGAAGTCAAGCGGTGGGACGCTCATCCTTTTACTGCCTGGAGTACTTCCCTTCGCAGATGCTAAGGACTGGAAATAACTTCCAATTCAGCTATACCTTCGAGGATGTACCTTTTCACAGCAGCTACGCTCACAGCCAGAGTTTGGATCGCTTGATGAATCCTCTTATTGATCAGTATCTGTACTACCTGAACAGAACGCAAGGAACAACCTCTGGAACAACCAACCAATCACGGCTGCTTTTTAGCCAGGCTGGGCCTCAGTCTATGTCTTTGCAGGCCAGAAATTGGCTACCTGGGCCCTGCTACCGGCAACAGAGACTTTCAAAGACTGCTAACGACAACAACAACAGTAACTTTCCTTGGACAGCGGCCAGCAAATATCATCTCAATGGCCGCGACTCGCTGGTGAATCCAGGACCAGCTATGGCCAGTCACAAGGACGATGAAGAAAAATTTTTCCCTATGCACGGCAATCTAATATTTGGCAAAGAAGGGACAACGGCAAGTAACGCAGAGATAATGTAATGATTACGGATGAAGAAGAGATTCGTACCACCAATCCTGTGGCAACAGAGCAGTATGGAACTGTGGCAAATAACTTGCAGAGCTCAAATACAGCTCCCACGACTGGAACTGTCAATCATCAGGGGGCCTTACCTGGCATGGTGTGGCAAGATCGTGACGTGTACCTTCAAGGACCTATCTGGGCAAAGATTCCTCACACGGATGGACACTTTCATCCTTCTCCTCTGATGGGAGGCTTTGGACTGAAACATCCGCCTCCTCAAATCATGATCAAAAATACTCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATCGTGGACTTACCGTGGATACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGACTACTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATGCATCGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGAATTCCAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATATTTGCTTATACAATCTTCCTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTGGAATTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGT TGGCCGATTCATTAATGCAG

[0338]

1 59 1 7214 DNA Adeno-associated virus misc_feature (1)..(7214) AAV2/4helper plasmid 1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacgcgcagcgtga 60 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttcccttcctttctcg 120 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctccctttagggttccgat 180 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggttcacgtagtg 240 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacgttctttaata 300 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctattcttttgatt 360 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatttaacaaaaat 420 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccattcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagctggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagtcacgacgttg 600 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaattggagctcca 660 ccgcggtggc ggccgctgct tatgtacgca gtagccatgg aaacgagataagataagaag 720 gacacggaga ccaaagttca actgaaacga ataaaccggt ttattgattaacaggttatt 780 acaggtggtg ggtgaggtag cgggtaccga tagccctagg ctcagtgtatttcccagccg 840 catcgggagc ccacaacaga gagttttgct gtccgtagtt ggaggtaaactggacctcgg 900 ggttccagcg tttggaccgc tccttctgga tctcccagtc aatctgcaccgacacctggc 960 cagtgctgta ctgagtaatg aaggagttta ccggagtaga gctgaaggtcgttgcaggat 1020 tcgcaggtac cggggtgttc ttgataaaaa tttgaggagg cgggtgtttcagcccaaacc 1080 caccaatcag cggtgagggg tgaaagtgtc catcggtatg aggaatcttggcccaaatgg 1140 gaccctggta gtaaatgtct ctgttttgcc agaccattcc aggcacggctcccaaggctg 1200 tcagtctgtc cacggtcggc aggttgctgt tgctctggtc accgccaggtaggttgcccc 1260 acatgtccgt atcggtggcg ttggtggctg ccagctcctc ctcagaggtgaagatcagag 1320 tcccgggtac ggtggccgtg ttgccgttct gtttaggccc cgcaaagatgagctggctgt 1380 tgctgaactt gctgtccgca ggtccagccg tggccattgg aggtccgggggtcagggcac 1440 tccatcttcc gtccagagtg ctgtgcgtct cgtatttgat gagactgtctgacccggtgg 1500 cagggatctt gtagttttga ttggcagtct ttgagaagcc ctgctgcttgattgaaggcc 1560 cgggcagcca gttcttttta aagttggaaa agttggtagg ccgcagcttggtaaagttgg 1620 tggtggcagt cccggcattc agggtggttc cggtggtggt cgattgcagtccccacaggt 1680 actggtcgat gagagggttc atcagccggt ccaggctctg gctgtgcgcgtacatcgagt 1740 ggaaaggcac cttctcaaaa ctgtacgtaa tttcaaagtt gttgccagtccgcagcatct 1800 gcgaaggaaa gtactccagg cagtagaagg catttctgtc agtctgttgctgcgaagtgt 1860 tgccggtcac cagtccacag tagccgtact ggggcaccat aaagacgtcgttgggaaaag 1920 gaggcaggct gccctcttga cccgcatcca tcacgtacgg cagttcgtacgacgagtccg 1980 caaagatctg aaccgtgctg gtaaggttat tagccaccgt tgtctcgccgttcgacgtcg 2040 tgacctcctt gacctggatg ttgaagattt tgacccgcat ggctttgggtcgcatgcccc 2100 agttgttgtt gatgagtcgc tgccagtcac gtggtgagaa gtggcagtggaagcggttga 2160 agtcaaagta tccccagggg gtggagaatc cgttgtaggt gttggactgcaggctctctc 2220 cgagtcgctt gtagaggtgg ttgttgtagg tgggcaagac ccaggttctggtgctggtgg 2280 tcgtgacgtg gccctcagac caggtggaat cgcaatgcca atcacccgaggcattaccca 2340 ctccatcggc accttgtccg ccctcgactg cagctccgcc agctgctgcacgcatctcac 2400 tgtcatcaga catggctccg gaagttgatc cctcaggggg tccgtcgcctgctccagttt 2460 cgtcttcgaa aacgagcttc tttttagccg gctgcttgcc ttttttgccgatacccgtgg 2520 aggagtcggg ctgctggggg gattcaatca acggtctctt ctttccaggagccgtctcac 2580 ccgcttgctc aaccagacca agaggttcaa gaaccctctt tttggcctggaagactgctc 2640 tgccgaggtt gcccccaaac gatgtgtcgc cctgaagccg ctgctggaactccgcgtcgg 2700 cgtggttgta cttgaggtag gggttgtcac cggccttgag ctgctggtcgtaggccttgt 2760 cgtgctcgag ggctgccgcg tccgctgcgt tgacgggttc ccccttgtcgagtccgttgc 2820 cgggtccgag gtatttgtaa cccggaagca caagaccccg agcgttgtcctgatgttgtt 2880 gatttgcctt gggtttaggg gctccaggtt gcagcgccca ccactctcgaacgccttcag 2940 agaggttgtc ctctagccaa tctggaaggt aaccgtcagt catatctggtttgagtcatt 3000 tattgttcca tgtcacagtc atccaagtcc acattggcca gttcgcaggccgagcaggcc 3060 acctcgggcg ccctccccat gatgtgatga atcggacaca gtttctgatacgtccgcttt 3120 ctgacgacag acacgggttg agattctgac acggggaagc actcggcacagtccatgacc 3180 ccgtgcgtga agcaaatgtc cacattctga ttcattctct cgcattgccggcagggaaaa 3240 agcatcagat tcatacccac gtgacgagaa catttgtttt ggtacctgtccgcgtagtcc 3300 accgaagctt ccgcgtctga cgtcgatggc tgcgcaactg actcgcgcacccgtttgggc 3360 tcacttatat ctgcgtcact gggggcgggt cttttcttgg ctccaccctttttgacgtag 3420 aattcatgct ccacctcaac cacgtgatcc tttgcccacc ggaaaaagtctttgacttcc 3480 tgcttggtga ccttcccaaa gtcatgatcc agacggcggg tgagttcaaatttgaacatc 3540 cggtcttgca acggctgctg gtgttcgaag gtcgttgagt tcccgtcaatcacggcgcac 3600 atgttggtgt tggaggtgac gatcacggga gtcgggtcta tctgggccgaggacttgcat 3660 ttctggtcca cgcgcacctt gcttcctccg agaatggctt tggccgactccacgaccttg 3720 gcggtcatct tcccctcctc ccaccagatc accatcttgt cgacacagtcgttgaaggga 3780 aagttctcat tggtccagtt tacgcacccg tagaagggca cagtgtgggctatggcctcc 3840 gcgatgttgg tcttcccggt agttgcaggc ccaaacagcc agatggtgttcctcttgccg 3900 aactttttcg tggcccatcc cagaaagacg gaagccgcat attggggatcgtacccgttt 3960 agttccaaaa ttttataaat ccgattgctg gaaatgtcct ccacgggctgctggcccacc 4020 aggtagtcgg gggcggtttt agtcaggctc ataatctttc ccgcattgtccaaggcagcc 4080 ttgatttggg accgcgagtt ggaggccgca ttgaaggaga tgtatgaggcctggtcctcc 4140 tggatccact gcttctccga ggtaatcccc ttgtccacga gccacccgaccagctccatg 4200 tacctggctg aagtttttga tctgatcacc ggcgcatcag aattgggattctgattctct 4260 ttgttctgct cctgcgtctg cgacacgtgc gtcagatgct gcgccaccaaccgtttacgc 4320 tccgtgagat tcaaacaggc gcttaaatac tgttccatat tagtccacgcccactggagc 4380 tcaggctggg ttttggggag caagtaattg gggatgtagc actcatccaccaccttgttc 4440 ccgcctccgg cgccatttct ggtctttgtg accgcgaacc agtttggcaaagtcggctcg 4500 atcccgcggt aaattctctg aatcagtttt tcgcgaatct gactcaggaaacgtcccaaa 4560 accatggatt tcaccccggt ggtttccacg agcacgtgca tgtggaagtagctctctccc 4620 ttctcaaatt gcacaaagaa aagggcctcc ggggccttac tcacacggcgccattccgtc 4680 agaaagtcgc gctgcagctt ctcggccacg gtcaggggtg cctgctcaatcagattcaga 4740 tccatgtcag aatctggcgg caactcccat tccttctcgg ccacccagttcacaaagctg 4800 tcagaaatgc cgggcagatg cccgtcaagg tcgctgggga ccttaatcacaatctcgtaa 4860 aaccccggca tggcggctgc gcgttcaaac ctcccgcttc aaaatggagaccctgcgtgc 4920 tcactcgggc ttaaataccc agcgtgacca catggtgtcg caaaatgtcgcaaaacactc 4980 acgtgacctc taatacagga ctctagcggt acccagcttt tgttccctttagtgagggtt 5040 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaattgttatccgct 5100 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggggtgcctaatg 5160 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagtcgggaaacct 5220 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtttgcgtattgg 5280 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggctgcggcgagc 5340 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcaggggataacgcagg 5400 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaaggccgcgttgct 5460 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgacgctcaagtca 5520 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctggaagctccct 5580 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcctttctcccttc 5640 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcggtgtaggtcgt 5700 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgctgcgccttatc 5760 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccactggcagcagc 5820 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagttcttgaagtg 5880 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctctgctgaagcc 5940 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaaccaccgctggtag 6000 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggatctcaagaaga 6060 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcacgttaagggat 6120 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaattaaaaatgaag 6180 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttaccaatgcttaat 6240 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttgcctgactccc 6300 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtgctgcaatgat 6360 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagccagccggaag 6420 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtctattaattgttg 6480 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttgttgccattgc 6540 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagctccggttccca 6600 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggttagctccttcgg 6660 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatggttatggcagc 6720 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtgactggtgagta 6780 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctcttgcccggcgtc 6840 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatcattggaaaacg 6900 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagttcgatgtaacc 6960 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgtttctgggtgagc 7020 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacggaaatgttgaat 7080 actcatactc ttcctttttc aatattattg aagcatttat cagggttattgtctcatgag 7140 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgcgcacatttcc 7200 ccgaaaagtg ccac 7214 2 8151 DNA Adeno-associated virusmisc_feature (1)..(8151) AAV2/4 helper plasmid 2 aattcccatc atcaataatataccttattt tggattgaag ccaatatgat aatgaggggg 60 tggagtttgt gacgtggcgcggggcgtggg aacggggcgg gtgacgtagt agtctctaga 120 gtcctgtatt agaggtcacgtgagtgtttt gcgacatttt gcgacaccat gtggtcacgc 180 tgggtattta agcccgagtgagcacgcagg gtctccattt tgaagcggga ggtttgaacg 240 cgcagccgcc atgccggggttttacgagat tgtgattaag gtccccagcg accttgacgg 300 gcatctgccc ggcatttctgacagctttgt gaactgggtg gccgagaagg aatgggagtt 360 gccgccagat tctgacatggatctgaatct gattgagcag gcacccctga ccgtggccga 420 gaagctgcag cgcgactttctgacggaatg gcgccgtgtg agtaaggccc cggaggccct 480 tttctttgtg caatttgagaagggagagag ctacttccac atgcacgtgc tcgtggaaac 540 caccggggtg aaatccatggttttgggacg tttcctgagt cagattcgcg aaaaactgat 600 tcagagaatt taccgcgggatcgagccgac tttgccaaac tggttcgcgg tcacaaagac 660 cagaaatggc gccggaggcgggaacaaggt ggtggatgag tgctacatcc ccaattactt 720 gctccccaaa acccagcctgagctccagtg ggcgtggact aatatggaac agtatttaag 780 cgcctgtttg aatctcacggagcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 840 gcagacgcag gagcagaacaaagagaatca gaatcccaat tctgatgcgc cggtgatcag 900 atcaaaaact tcagccaggtacatggagct ggtcgggtgg ctcgtggaca aggggattac 960 ctcggagaag cagtggatccaggaggacca ggcctcatac atctccttca atgcggcctc 1020 caactcgcgg tcccaaatcaaggctgcctt ggacaatgcg ggaaagatta tgagcctgac 1080 taaaaccgcc cccgactacctggtgggcca gcagcccgtg gaggacattt ccagcaatcg 1140 gatttataaa attttggaactaaacgggta cgatccccaa tatgcggctt ccgtctttct 1200 gggatgggcc acgaaaaagttcggcaagag gaacaccatc tggctgtttg ggcctgcaac 1260 taccgggaag accaacatcgcggaggccat agcccacact gtgcccttct acgggtgcgt 1320 aaactggacc aatgagaactttcccttcaa cgactgtgtc gacaagatgg tgatctggtg 1380 ggaggagggg aagatgaccgccaaggtcgt ggagtcggcc aaagccattc tcggaggaag 1440 caaggtgcgc gtggaccagaaatgcaagtc ctcggcccag atagacccga ctcccgtgat 1500 cgtcacctcc aacaccaacatgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1560 ccagcagccg ttgcaagaccggatgttcaa atttgaactc acccgccgtc tggatcatga 1620 ctttgggaag gtcaccaagcaggaagtcaa agactttttc cggtgggcaa aggatcacgt 1680 ggttgaggtg gagcatgaattctacgtcaa aaagggtgga gccaagaaaa gacccgcccc 1740 cagtgacgca gatataagtgagcccaaacg ggtgcgcgag tcagttgcgc agccatcgac 1800 gtcagacgcg gaagcttcgatcaactacgc agacaggtac caaaacaaat gttctcgtca 1860 cgtgggcatg aatctgatgctgtttccctg cagacaatgc gagagaatga atcagaattc 1920 aaatatctgc ttcactcacggacagaaaga ctgtttagag tgctttcccg tgtcagaatc 1980 tcaacccgtt tctgtcgtcaaaaaggcgta tcagaaactg tgctacattc atcatatcat 2040 gggaaaggtg ccagacgcttgcactgcctg cgatctggtc aatgtggatt tggatgactg 2100 catctttgaa caataaatgatttaaatcag gtatggctgc cgatggttat cttccagatt 2160 ggctcgagga cactctctctgaaggaataa gacagtggtg gaagctcaaa cctggcccac 2220 caccaccaaa gcccgcagagcggcataagg acgacagcag gggtcttgtg cttcctgggt 2280 acaagtacct cggacccttcaacggactcg acaagggaga gccggtcaac gaggcagacg 2340 ccgcggccct cgagcacgacaaggcctacg accagcagct caaggccggt gacaacccct 2400 acctcaagta caaccacgccgacgcggagt tccagcagcg gcttcagggc gacacatcgt 2460 ttgggggcaa cctcggcagagcagtcttcc aggccaaaaa gagggttctt gaacctcttg 2520 gtctggttga gcaagcgggtgagacggctc ctggaaagaa gagaccgttg attgaatccc 2580 cccagcagcc cgactcctccacgggtatcg gcaaaaaagg caagcagccg gctaaaaaga 2640 agctcgtttt cgaagacgaaactggagcag gcgacggacc ccctgaggga tcaacttccg 2700 gagccatgtc tgatgacagtgagatgcgtg cagcagctgg cggagctgca gtcgagggcg 2760 gacaaggtgc cgatggagtgggtaatgcct cgggtgattg gcattgcgat tccacctggt 2820 ctgagggcca cgtcacgaccaccagcacca gaacctgggt cttgcccacc tacaacaacc 2880 acctctacaa gcgactcggagagagcctgc agtccaacac ctacaacgga ttctccaccc 2940 cctggggata ctttgacttcaaccgcttcc actgccactt ctcaccacgt gactggcagc 3000 gactcatcaa caacaactggggcatgcgac ccaaagccat gcgggtcaaa atcttcaaca 3060 tccaggtcaa ggaggtcacgacgtcgaacg gcgagacaac ggtggctaat aaccttacca 3120 gcacggttca gatctttgcggactcgtcgt acgaactgcc gtacgtcctc ggctcggcgc 3180 atcaaggatg cctcccgccgttcccagcag acgtcttcat ggtgccacag tatggatacc 3240 tcaccctgaa caacgggagtcaggcagtag gacgctcttc attttactgc ctggagtact 3300 ttccttctca gatgctgcgtaccggaaaca actttacctt cagctacact tttgaggacg 3360 ttcctttcca cagcagctacgctcacagcc agagtctgga ccgtctcatg aatcctctca 3420 tcgaccagta cctgtattacttgagcagaa caaacactcc aagtggaacc accacgcagt 3480 caaggcttca gttttctcaggccggagcga gtgacattcg ggaccagtct aggaactggc 3540 ttcctggacc ctgttaccgccagcagcgag tatcaaagac atctgcggat aacaacaaca 3600 gtgaatactc gtggactggagctaccaagt accacctcaa tggcagagac tctctggtga 3660 atccgggccc ggccatggcaagccacaagg acgatgaaga aaagtttttt cctcagagcg 3720 gggttctcat ctttgggaagcaaggctcag agaaaacaaa tgtgaacatt gaaaaggtca 3780 tgattacaga cgaagaggaaatcggaacaa ccaatcccgt ggctacggag cagtatggtt 3840 ctgtatctac caacctccagagaggcaaca gacaagcagc taccgcagat gtcaacacac 3900 aaggcgttct tccaggcatggtctggcagg acagagatgt gtaccttcag gggcccatct 3960 gggcaaagat tccacacacggacggacatt ttcacccctc tcccctcatg ggtggattcg 4020 gacttaaaca ccctcctccacagattctca tcaagaacac cccggtacct gcgaatcctt 4080 cgaccacctt cagtgcggcaaagtttgctt ccttcatcac acagtactcc acgggacagg 4140 tcagcgtgga gatcgagtgggagctgcaga aggaaaacag caaacgctgg aatcccgaaa 4200 ttcagtacac ttccaactacaacaagtctg ttaatcgtgg acttaccgtg gatactaatg 4260 gcgtgtattc agagcctcgccccattggca ccagatacct gactcgtaat ctgtaattgc 4320 ttgttaatca ataaaccgtttaattcgttt cagttgaact ttggtctctg cgtatttctt 4380 tcttatctag tttccatgctctagactact acgtcacccg ccccgttccc acgccccgcg 4440 ccacgtcaca aactccaccccctcattatc atattggctt caatccaaaa taaggtatat 4500 tattgatgat gcatcgctggcgtaatagcg aagaggcccg caccgatcgc ccttcccaac 4560 agttgcgcag cctgaatggcgaatggaatt ccagacgatt gagcgtcaaa atgtaggtat 4620 ttccatgagc gtttttcctgttgcaatggc tggcggtaat attgttctgg atattaccag 4680 caaggccgat agtttgagttcttctactca ggcaagtgat gttattacta atcaaagaag 4740 tattgcgaca acggttaatttgcgtgatgg acagactctt ttactcggtg gcctcactga 4800 ttataaaaac acttctcaggattctggcgt accgttcctg tctaaaatcc ctttaatcgg 4860 cctcctgttt agctcccgctctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 4920 agcaaccata gtacgcgccctgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 4980 gcagcgtgac cgctacacttgccagcgccc tagcgcccgc tcctttcgct ttcttccctt 5040 cctttctcgc cacgttcgccggctttcccc gtcaagctct aaatcggggg ctccctttag 5100 ggttccgatt tagtgctttacggcacctcg accccaaaaa acttgattag ggtgatggtt 5160 cacgtagtgg gccatcgccctgatagacgg tttttcgccc tttgacgttg gagtccacgt 5220 tctttaatag tggactcttgttccaaactg gaacaacact caaccctatc tcggtctatt 5280 cttttgattt ataagggattttgccgattt cggcctattg gttaaaaaat gagctgattt 5340 aacaaaaatt taacgcgaattttaacaaaa tattaacgtt tacaatttaa atatttgctt 5400 atacaatctt cctgtttttggggcttttct gattatcaac cggggtacat atgattgaca 5460 tgctagtttt acgattaccgttcatcgatt ctcttgtttg ctccagactc tcaggcaatg 5520 acctgatagc ctttgtagagacctctcaaa aatagctacc ctctccggca tgaatttatc 5580 agctagaacg gttgaatatcatattgatgg tgatttgact gtctccggcc tttctcaccc 5640 gtttgaatct ttacctacacattactcagg cattgcattt aaaatatatg agggttctaa 5700 aaatttttat ccttgcgttgaaataaaggc ttctcccgca aaagtattac agggtcataa 5760 tgtttttggt acaaccgatttagctttatg ctctgaggct ttattgctta attttgctaa 5820 ttctttgcct tgcctgtatgatttattgga tgttggaatt cctgatgcgg tattttctcc 5880 ttacgcatct gtgcggtatttcacaccgca tatggtgcac tctcagtaca atctgctctg 5940 atgccgcata gttaagccagccccgacacc cgccaacacc cgctgacgcg ccctgacggg 6000 cttgtctgct cccggcatccgcttacagac aagctgtgac cgtctccggg agctgcatgt 6060 gtcagaggtt ttcaccgtcatcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 6120 tatttttata ggttaatgtcatgataataa tggtttctta gacgtcaggt ggcacttttc 6180 ggggaaatgt gcgcggaacccctatttgtt tatttttcta aatacattca aatatgtatc 6240 cgctcatgag acaataaccctgataaatgc ttcaataata ttgaaaaagg aagagtatga 6300 gtattcaaca tttccgtgtcgcccttattc ccttttttgc ggcattttgc cttcctgttt 6360 ttgctcaccc agaaacgctggtgaaagtaa aagatgctga agatcagttg ggtgcacgag 6420 tgggttacat cgaactggatctcaacagcg gtaagatcct tgagagtttt cgccccgaag 6480 aacgttttcc aatgatgagcacttttaaag ttctgctatg tggcgcggta ttatcccgta 6540 ttgacgccgg gcaagagcaactcggtcgcc gcatacacta ttctcagaat gacttggttg 6600 agtactcacc agtcacagaaaagcatctta cggatggcat gacagtaaga gaattatgca 6660 gtgctgccat aaccatgagtgataacactg cggccaactt acttctgaca acgatcggag 6720 gaccgaagga gctaaccgcttttttgcaca acatggggga tcatgtaact cgccttgatc 6780 gttgggaacc ggagctgaatgaagccatac caaacgacga gcgtgacacc acgatgcctg 6840 tagcaatggc aacaacgttgcgcaaactat taactggcga actacttact ctagcttccc 6900 ggcaacaatt aatagactggatggaggcgg ataaagttgc aggaccactt ctgcgctcgg 6960 cccttccggc tggctggtttattgctgata aatctggagc cggtgagcgt gggtctcgcg 7020 gtatcattgc agcactggggccagatggta agccctcccg tatcgtagtt atctacacga 7080 cggggagtca ggcaactatggatgaacgaa atagacagat cgctgagata ggtgcctcac 7140 tgattaagca ttggtaactgtcagaccaag tttactcata tatactttag attgatttaa 7200 aacttcattt ttaatttaaaaggatctagg tgaagatcct ttttgataat ctcatgacca 7260 aaatccctta acgtgagttttcgttccact gagcgtcaga ccccgtagaa aagatcaaag 7320 gatcttcttg agatcctttttttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 7380 cgctaccagc ggtggtttgtttgccggatc aagagctacc aactcttttt ccgaaggtaa 7440 ctggcttcag cagagcgcagataccaaata ctgtccttct agtgtagccg tagttaggcc 7500 accacttcaa gaactctgtagcaccgccta catacctcgc tctgctaatc ctgttaccag 7560 tggctgctgc cagtggcgataagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 7620 cggataaggc gcagcggtcgggctgaacgg ggggttcgtg cacacagccc agcttggagc 7680 gaacgaccta caccgaactgagatacctac agcgtgagct atgagaaagc gccacgcttc 7740 ccgaagggag aaaggcggacaggtatccgg taagcggcag ggtcggaaca ggagagcgca 7800 cgagggagct tccagggggaaacgcctggt atctttatag tcctgtcggg tttcgccacc 7860 tctgacttga gcgtcgatttttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 7920 ccagcaacgc ggcctttttacggttcctgg ccttttgctg gccttttgct cacatgttct 7980 ttcctgcgtt atcccctgattctgtggata accgtattac cgcctttgag tgagctgata 8040 ccgctcgccg cagccgaacgaccgagcgca gcgagtcagt gagcgaggaa gcggaagagc 8100 gcccaatacg caaaccgcctctccccgcgc gttggccgat tcattaatgc a 8151 3 2271 DNA Adeno-associatedvirus misc_feature (1)..(2271) B19/AAV chimeric capsid coding sequence 3atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac act ctc tct 48 MetAla Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15gaa gga ata aga cag tgg tgg aag ctc aaa cct ggc cca cca cca cca 96 GluGly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 aagccc gca gag cgg cat aag gac gac agc agg ggt ctt gtg ctt cct 144 Lys ProAla Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 ggg tacaag tac ctc gga ccc ttc aac gga ctc gac aag gga gag ccg 192 Gly Tyr LysTyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 gtc aac gaggca gac gcc gcg gcc ctc gag cac gac aaa gcc tac gac 240 Val Asn Glu AlaAsp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 cgg cag ctcgac agc gga gac aac ccg tac ctc aag tac aac cac gcc 288 Arg Gln Leu AspSer Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 gac gcg gag tttcag gag cgc ctt aaa gaa gat acg tct ttt ggg ggc 336 Asp Ala Glu Phe GlnGlu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 aac ctc gga cgagca gtc ttc cag gcg aaa aag agg gtt ctt gaa cct 384 Asn Leu Gly Arg AlaVal Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 ctg ggc ctg gttgag gaa cct gtt aag acg gct ccg gga aaa aag agg 432 Leu Gly Leu Val GluGlu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 ccg gta gag cactct cct gtg gag cca gac tcc tcc tcg gga acc gga 480 Pro Val Glu His SerPro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 aag gcg ggccag cag cct gca aga aaa aga ttg aat ttt ggt cag act 528 Lys Ala Gly GlnGln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 gga gac gcagac tca gta cct gac ccc cag cct ctc gga cag cca cca 576 Gly Asp Ala AspSer Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 gca gcc ccctct ggt ctg gga act aat acg atg act tca gtt aat tct 624 Ala Ala Pro SerGly Leu Gly Thr Asn Thr Met Thr Ser Val Asn Ser 195 200 205 gca gaa gccagc act ggt gca gga ggg ggg ggc agt aat tct gtc aaa 672 Ala Glu Ala SerThr Gly Ala Gly Gly Gly Gly Ser Asn Ser Val Lys 210 215 220 agc atg tggagt gag ggg gcc act ttt agt gct aac tct gta act tgt 720 Ser Met Trp SerGlu Gly Ala Thr Phe Ser Ala Asn Ser Val Thr Cys 225 230 235 240 aca ttttcc aga cag ttt tta att cca tat gac cca gag cac cat tat 768 Thr Phe SerArg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His Tyr 245 250 255 aag gtgttt tct ccc gca gcg agt agc tgc cac aat gcc agt gga aag 816 Lys Val PheSer Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly Lys 260 265 270 gag gcaaag gtt tgc acc atc agt ccc ata atg gga tac tca acc cca 864 Glu Ala LysVal Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr Pro 275 280 285 tgg agatat tta gat ttt aat gct tta aat tta ttt ttt tca cct tta 912 Trp Arg TyrLeu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro Leu 290 295 300 gag tttcag cac tta att gaa aat tat gga agt ata gct cct gat gct 960 Glu Phe GlnHis Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp Ala 305 310 315 320 ttaact gta acc ata tca gaa att gct gtt aag gat gtt aca gac aaa 1008 Leu ThrVal Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp Lys 325 330 335 actgga ggg ggg gta cag gtt act gac agc act aca ggg cgc cta tgc 1056 Thr GlyGly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg Leu Cys 340 345 350 atgtta gta gac cat gaa tac aag tac cca tat gtg tta ggg caa ggt 1104 Met LeuVal Asp His Glu Tyr Lys Tyr Pro Tyr Val Leu Gly Gln Gly 355 360 365 caggat act tta gcc cca gaa ctt cct att tgg gta tac ttt ccc cct 1152 Gln AspThr Leu Ala Pro Glu Leu Pro Ile Trp Val Tyr Phe Pro Pro 370 375 380 caatat gct tac tta aca gta gga gat gtt aac aca caa gga att tct 1200 Gln TyrAla Tyr Leu Thr Val Gly Asp Val Asn Thr Gln Gly Ile Ser 385 390 395 400gga gac agc aaa aaa tta gca agt gaa gaa tca gca ttt tat gtt ttg 1248 GlyAsp Ser Lys Lys Leu Ala Ser Glu Glu Ser Ala Phe Tyr Val Leu 405 410 415gaa cac agt tct ttt cag ctt tta ggt aca gga ggt aca gca act atg 1296 GluHis Ser Ser Phe Gln Leu Leu Gly Thr Gly Gly Thr Ala Thr Met 420 425 430tct tat aag ttt cct cca gtg ccc cca gaa aat tta gag ggc tgc agt 1344 SerTyr Lys Phe Pro Pro Val Pro Pro Glu Asn Leu Glu Gly Cys Ser 435 440 445caa cac ttt tat gaa atg tac aat ccc tta tac gga tcc cgc tta ggg 1392 GlnHis Phe Tyr Glu Met Tyr Asn Pro Leu Tyr Gly Ser Arg Leu Gly 450 455 460gtt cct gac aca tta gga ggt gac cca aaa ttt aga tct tta aca cat 1440 ValPro Asp Thr Leu Gly Gly Asp Pro Lys Phe Arg Ser Leu Thr His 465 470 475480 gaa gac cat gca att cag ccc caa aac ttc atg cca ggg cca cta gta 1488Glu Asp His Ala Ile Gln Pro Gln Asn Phe Met Pro Gly Pro Leu Val 485 490495 aac tca gtg tct aca aag gag gga gac agc tct aat act gga gct gga 1536Asn Ser Val Ser Thr Lys Glu Gly Asp Ser Ser Asn Thr Gly Ala Gly 500 505510 aaa gcc tta aca ggc ctt agc aca ggt acc tct caa aac act aga ata 1584Lys Ala Leu Thr Gly Leu Ser Thr Gly Thr Ser Gln Asn Thr Arg Ile 515 520525 tcc tta cgc cct ggg cca gtg tct cag cca tac cac cac tgg gac aca 1632Ser Leu Arg Pro Gly Pro Val Ser Gln Pro Tyr His His Trp Asp Thr 530 535540 gat aaa tat gtc aca gga ata aat gcc att tct cat ggt cag acc act 1680Asp Lys Tyr Val Thr Gly Ile Asn Ala Ile Ser His Gly Gln Thr Thr 545 550555 560 tat ggt aac gct gaa gac aaa gag tat cag caa gga gtg ggt aga ttt1728 Tyr Gly Asn Ala Glu Asp Lys Glu Tyr Gln Gln Gly Val Gly Arg Phe 565570 575 cca aat gaa aaa gaa cag cta aaa cag tta cag ggt tta aac atg cac1776 Pro Asn Glu Lys Glu Gln Leu Lys Gln Leu Gln Gly Leu Asn Met His 580585 590 acc tac ttt ccc aat aaa gga acc cag caa tat aca gat caa att gag1824 Thr Tyr Phe Pro Asn Lys Gly Thr Gln Gln Tyr Thr Asp Gln Ile Glu 595600 605 cgc ccc cta atg gtg ggt tct gta tgg aac aga aga gcc ctt cac tat1872 Arg Pro Leu Met Val Gly Ser Val Trp Asn Arg Arg Ala Leu His Tyr 610615 620 gaa agc cag ctg tgg agt aaa att cca aat tta gat gac agt ttt aaa1920 Glu Ser Gln Leu Trp Ser Lys Ile Pro Asn Leu Asp Asp Ser Phe Lys 625630 635 640 act cag ttt gca gcc tta gga gga tgg ggt ttg cat cag cca cctcct 1968 Thr Gln Phe Ala Ala Leu Gly Gly Trp Gly Leu His Gln Pro Pro Pro645 650 655 caa ata ttt tta aaa ata tta cca caa agt ggg cca att gga ggtatt 2016 Gln Ile Phe Leu Lys Ile Leu Pro Gln Ser Gly Pro Ile Gly Gly Ile660 665 670 aaa tca atg gga att act acc tta gtt cag tat gcc gtg gga attatg 2064 Lys Ser Met Gly Ile Thr Thr Leu Val Gln Tyr Ala Val Gly Ile Met675 680 685 aca gta act atg aca ttt aaa ttg ggg ccc cgt aaa gct acg ggacgg 2112 Thr Val Thr Met Thr Phe Lys Leu Gly Pro Arg Lys Ala Thr Gly Arg690 695 700 tgg aat cct caa cct gga gta tat ccc ccg cac gca gca ggt cattta 2160 Trp Asn Pro Gln Pro Gly Val Tyr Pro Pro His Ala Ala Gly His Leu705 710 715 720 cca tat gta cta tat gac ccc aca gct aca gat gca aaa caacac cac 2208 Pro Tyr Val Leu Tyr Asp Pro Thr Ala Thr Asp Ala Lys Gln HisHis 725 730 735 aga cat gga tat gaa aag cct gaa gaa ttg tgg aca gcc aaaagc cgt 2256 Arg His Gly Tyr Glu Lys Pro Glu Glu Leu Trp Thr Ala Lys SerArg 740 745 750 gtg cac cca ttg taa 2271 Val His Pro Leu 755 4 756 PRTAdeno-associated virus misc_feature (1)..(2271) B19/AAV chimeric capsidcoding sequence 4 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu AspThr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro GlyPro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg GlyLeu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu AspLys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His AspLys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr LeuLys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu AspThr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala LysLys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val LysThr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val GluPro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln ProAla Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp SerVal Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro SerGly Leu Gly Thr Asn Thr Met Thr Ser Val Asn Ser 195 200 205 Ala Glu AlaSer Thr Gly Ala Gly Gly Gly Gly Ser Asn Ser Val Lys 210 215 220 Ser MetTrp Ser Glu Gly Ala Thr Phe Ser Ala Asn Ser Val Thr Cys 225 230 235 240Thr Phe Ser Arg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His Tyr 245 250255 Lys Val Phe Ser Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly Lys 260265 270 Glu Ala Lys Val Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr Pro275 280 285 Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser ProLeu 290 295 300 Glu Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala ProAsp Ala 305 310 315 320 Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys AspVal Thr Asp Lys 325 330 335 Thr Gly Gly Gly Val Gln Val Thr Asp Ser ThrThr Gly Arg Leu Cys 340 345 350 Met Leu Val Asp His Glu Tyr Lys Tyr ProTyr Val Leu Gly Gln Gly 355 360 365 Gln Asp Thr Leu Ala Pro Glu Leu ProIle Trp Val Tyr Phe Pro Pro 370 375 380 Gln Tyr Ala Tyr Leu Thr Val GlyAsp Val Asn Thr Gln Gly Ile Ser 385 390 395 400 Gly Asp Ser Lys Lys LeuAla Ser Glu Glu Ser Ala Phe Tyr Val Leu 405 410 415 Glu His Ser Ser PheGln Leu Leu Gly Thr Gly Gly Thr Ala Thr Met 420 425 430 Ser Tyr Lys PhePro Pro Val Pro Pro Glu Asn Leu Glu Gly Cys Ser 435 440 445 Gln His PheTyr Glu Met Tyr Asn Pro Leu Tyr Gly Ser Arg Leu Gly 450 455 460 Val ProAsp Thr Leu Gly Gly Asp Pro Lys Phe Arg Ser Leu Thr His 465 470 475 480Glu Asp His Ala Ile Gln Pro Gln Asn Phe Met Pro Gly Pro Leu Val 485 490495 Asn Ser Val Ser Thr Lys Glu Gly Asp Ser Ser Asn Thr Gly Ala Gly 500505 510 Lys Ala Leu Thr Gly Leu Ser Thr Gly Thr Ser Gln Asn Thr Arg Ile515 520 525 Ser Leu Arg Pro Gly Pro Val Ser Gln Pro Tyr His His Trp AspThr 530 535 540 Asp Lys Tyr Val Thr Gly Ile Asn Ala Ile Ser His Gly GlnThr Thr 545 550 555 560 Tyr Gly Asn Ala Glu Asp Lys Glu Tyr Gln Gln GlyVal Gly Arg Phe 565 570 575 Pro Asn Glu Lys Glu Gln Leu Lys Gln Leu GlnGly Leu Asn Met His 580 585 590 Thr Tyr Phe Pro Asn Lys Gly Thr Gln GlnTyr Thr Asp Gln Ile Glu 595 600 605 Arg Pro Leu Met Val Gly Ser Val TrpAsn Arg Arg Ala Leu His Tyr 610 615 620 Glu Ser Gln Leu Trp Ser Lys IlePro Asn Leu Asp Asp Ser Phe Lys 625 630 635 640 Thr Gln Phe Ala Ala LeuGly Gly Trp Gly Leu His Gln Pro Pro Pro 645 650 655 Gln Ile Phe Leu LysIle Leu Pro Gln Ser Gly Pro Ile Gly Gly Ile 660 665 670 Lys Ser Met GlyIle Thr Thr Leu Val Gln Tyr Ala Val Gly Ile Met 675 680 685 Thr Val ThrMet Thr Phe Lys Leu Gly Pro Arg Lys Ala Thr Gly Arg 690 695 700 Trp AsnPro Gln Pro Gly Val Tyr Pro Pro His Ala Ala Gly His Leu 705 710 715 720Pro Tyr Val Leu Tyr Asp Pro Thr Ala Thr Asp Ala Lys Gln His His 725 730735 Arg His Gly Tyr Glu Lys Pro Glu Glu Leu Trp Thr Ala Lys Ser Arg 740745 750 Val His Pro Leu 755 5 8179 DNA Adeno-associated virus 5aattcccatc atcaataata taccttattt tggattgaag ccaatatgat aatgaggggg 60tggagtttgt gacgtggcgc ggggcgtggg aacggggcgg gtgacgtagt agtctctaga 120gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat gtggtcacgc 180tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga ggtttgaacg 240cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacgg 300gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt 360gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga 420gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct 480tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac 540caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat 600tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac 660cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt 720gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag 780cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 840gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag 900atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac 960ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc 1020caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac 1080taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg 1140gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct 1200gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac 1260taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt 1320aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg 1380ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag 1440caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat 1500cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1560ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga 1620ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt 1680ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc 1740cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac 1800gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca 1860cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc 1920aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc 1980tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat 2040gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg 2100catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt 2160ggctcgagga cactctctct gaaggaataa gacagtggtg gaagctcaaa cctggcccac 2220caccaccaaa gcccgcagag cggcataagg acgacagcag gggtcttgtg cttcctgggt 2280acaagtacct cggacccttc aacggactcg acaagggaga gccggtcaac gaggcagacg 2340ccgcggccct cgagcacgac aaagcctacg accggcagct cgacagcgga gacaacccgt 2400acctcaagta caaccacgcc gacgcggagt ttcaggagcg ccttaaagaa gatacgtctt 2460ttgggggcaa cctcggacga gcagtcttcc aggcgaaaaa gagggttctt gaacctctgg 2520gcctggttga ggaacctgtt aagacggctc cgggaaaaaa gaggccggta gagcactctc 2580ctgtggagcc agactcctcc tcgggaaccg gaaaggcggg ccagcagcct gcaagaaaaa 2640gattgaattt tggtcagact ggagacgcag actcagtacc tgacccccag cctctcggac 2700agccaccagc agccccctct ggtctgggaa ctaatacgat ggctacaggc agtggcgcac 2760caatggcaga caataacgag ggcgccgacg gagtgggtaa ttcctccgga aattggcatt 2820gcgattccac atggatgggc gacagagtca tcaccaccag cacccgaacc tgggccctgc 2880ccacctacaa caaccacctc tacaaacaaa tttccagcca atcaggagcc tcgaacgaca 2940atcactactt tggctacagc accccttggg ggtattttga cttcaacaga ttccactgcc 3000acttttcacc acgtgactgg caaagactca tcaacaacaa ctggggattc cgacccaaga 3060gactcaactt caagctcttt aacattcaag tcaaagaggt cacgcagaat gacggtacga 3120cgacgattgc caataacctt accagcacgg ttcaggtgtt tactgactcg gagtaccagc 3180tcccgtacgt gctcgggtcg gcgcaccaag gctgtctccc gccgtttcca gcggacgtct 3240tcatggtccc tcagtatgga tacctcaccc tgaacaacgg aagtcaagcg gtgggacgct 3300catcctttta ctgcctggag tacttccctt cgcagatgct aaggactgga aataacttcc 3360aattcagcta taccttcgag gatgtacctt ttcacagcag ctacgctcac agccagagtt 3420tggatcgctt gatgaatcct cttattgatc agtatctgta ctacctgaac agaacgcaag 3480gaacaacctc tggaacaacc aaccaatcac ggctgctttt tagccaggct gggcctcagt 3540ctatgtcttt gcaggccaga aattggctac ctgggccctg ctaccggcaa cagagacttt 3600caaagactgc taacgacaac aacaacagta actttccttg gacagcggcc agcaaatatc 3660atctcaatgg ccgcgactcg ctggtgaatc caggaccagc tatggccagt cacaaggacg 3720atgaagaaaa atttttccct atgcacggca atctaatatt tggcaaagaa gggacaacgg 3780caagtaacgc agaattagat aatgtaatga ttacggatga agaagagatt cgtaccacca 3840atcctgtggc aacagagcag tatggaactg tggcaaataa cttgcagagc tcaaatacag 3900ctcccacgac tggaactgtc aatcatcagg gggccttacc tggcatggtg tggcaagatc 3960gtgacgtgta ccttcaagga cctatctggg caaagattcc tcacacggat ggacactttc 4020atccttctcc tctgatggga ggctttggac tgaaacatcc gcctcctcaa atcatgatca 4080aaaatactcc ggtacctgcg aatccttcga ccaccttcag tgcggcaaag tttgcttcct 4140tcatcacaca gtactccacg ggacaggtca gcgtggagat cgagtgggag ctgcagaagg 4200aaaacagcaa acgctggaat cccgaaattc agtacacttc caactacaac aagtctgtta 4260atcgtggact taccgtggat actaatggcg tgtattcaga gcctcgcccc attggcacca 4320gatacctgac tcgtaatctg taattgcttg ttaatcaata aaccgtttaa ttcgtttcag 4380ttgaactttg gtctctgcgt atttctttct tatctagttt ccatgctcta gactactacg 4440tcacccgccc cgttcccacg ccccgcgcca cgtcacaaac tccaccccct cattatcata 4500ttggcttcaa tccaaaataa ggtatattat tgatgatgca tcgctggcgt aatagcgaag 4560aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaattcca 4620gacgattgag cgtcaaaatg taggtatttc catgagcgtt tttcctgttg caatggctgg 4680cggtaatatt gttctggata ttaccagcaa ggccgatagt ttgagttctt ctactcaggc 4740aagtgatgtt attactaatc aaagaagtat tgcgacaacg gttaatttgc gtgatggaca 4800gactctttta ctcggtggcc tcactgatta taaaaacact tctcaggatt ctggcgtacc 4860gttcctgtct aaaatccctt taatcggcct cctgtttagc tcccgctctg attctaacga 4920ggaaagcacg ttatacgtgc tcgtcaaagc aaccatagta cgcgccctgt agcggcgcat 4980taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 5040cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 5100aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 5160ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 5220ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 5280caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 5340cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 5400taacgtttac aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat 5460tatcaaccgg ggtacatatg attgacatgc tagttttacg attaccgttc atcgattctc 5520ttgtttgctc cagactctca ggcaatgacc tgatagcctt tgtagagacc tctcaaaaat 5580agctaccctc tccggcatga atttatcagc tagaacggtt gaatatcata ttgatggtga 5640tttgactgtc tccggccttt ctcacccgtt tgaatcttta cctacacatt actcaggcat 5700tgcatttaaa atatatgagg gttctaaaaa tttttatcct tgcgttgaaa taaaggcttc 5760tcccgcaaaa gtattacagg gtcataatgt ttttggtaca accgatttag ctttatgctc 5820tgaggcttta ttgcttaatt ttgctaattc tttgccttgc ctgtatgatt tattggatgt 5880tggaattcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 5940ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 6000caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 6060ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 6120cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 6180tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 6240ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 6300aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 6360tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 6420atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 6480agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 6540tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 6600tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 6660atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 6720ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 6780tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 6840acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 6900ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 6960aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 7020ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 7080cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 7140gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 7200actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 7260agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 7320cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 7380tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 7440agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 7500tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 7560acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 7620ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 7680gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 7740gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 7800gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 7860tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 7920caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 7980tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 8040gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 8100agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 8160ggccgattca ttaatgcag 8179 6 17 DNA Artificial sequence Syntheticoligonucleotide 6 aattcgccgg cgatatc 17 7 16 DNA Artificial sequenceSynthetic oligonucleotide 7 tcgagatatc gccggc 16 8 12 DNA Artificialsequence Synthetic oligonucleotide 8 ggcgatatcg cc 12 9 48 DNAArtificial sequence Synthetic oligonucleotide 9 gctagcggcg gacaccatcaccaccaccat caccacggcg gaagcgct 48 10 48 DNA Artificial sequenceSynthetic oligonucleotide 10 agcgcttccg ccgtggtgat ggtggtggtg atggtgtccgccgctagc 48 11 51 DNA Artificial sequence Synthetic oligonucleotide 11acgctagcgg cggacaccat caccaccacc atcaccacgg cggaagcgct t 51 12 51 DNAArtificial sequence Synthetic oligonucleotide 12 aagcgcttcc gccgtggtgatggtggtggt gatggtgtcc gccgctagcg t 51 13 51 DNA Artificial sequenceSynthetic oligonucleotide 13 gggttccgga gggcaccacc atcaccacca ccatcacggaggcgccagcg a 51 14 51 DNA Artificial sequence Synthetic oligonucleotide14 tcgctggcgc ctccgtgatg gtggtggtga tggtggtgcc ctccggaacc c 51 15 60 DNAArtificial sequence Synthetic oligonucleotide 15 gccggatccg gcggcggctccagacccccc ggcttcagcc ccttcagatc cggcggcgcc 60 16 60 DNA Artificialsequence Synthetic oligonucleotide 16 ggcgccgccg gatctgaagg ggctgaagccggggggtctg gagccgccgc cggatccggc 60 17 69 DNA Artificial sequenceSynthetic oligonucleotide 17 gaggttcatg tgactgcggg ggaagacccc ctggcttcagcccattcaga ggtggctgct 60 tctgtggcg 69 18 69 DNA Artificial sequenceSynthetic oligonucleotide 18 cgccacagaa gcagccacct ctgaatgggc tgaagccagggggtcttccc ccgcagtcac 60 atgaacctc 69 19 69 DNA Artificial sequenceSynthetic oligonucleotide 19 aggttcatgt gactgcgggg gaagaccccc tggcttcagcccattcagag gtggctgctt 60 ctgtggcgg 69 20 69 DNA Artificial sequenceSynthetic oligonucleotide 20 ccgccacaga agcagccacc tctgaatggg ctgaagccagggggtcttcc cccgcagtca 60 catgaacct 69 21 36 DNA Artificial sequenceSynthetic oligonucleotide 21 ggatcctgcg actgcagggg cgattgtttc tgcggc 3622 36 DNA Artificial sequence Synthetic oligonucleotide 22 gccgcagaaacaatcgcccc tgcagtcgca ggatcc 36 23 36 DNA Artificial sequence Syntheticoligonucleotide 23 gatcctcgga ctgcaggggc gattgtttct gcggcg 36 24 36 DNAArtificial sequence Synthetic oligonucleotide 24 cgccgcagaa acaatcgcccctgcagtcgc aggatc 36 25 36 DNA Artificial sequence Syntheticoligonucleotide 25 aggatcctgc gactgcaggg gcgattgttt ctgcgg 36 26 36 DNAArtificial sequence Synthetic oligonucleotide 26 ccgcagaaac aatcgcccctgcagtcgcag gatcct 36 27 63 DNA Artificial sequence Syntheticoligonucleotide 27 aggttcatgt gactgcgggg gaaagaagaa gaagaagaagaagggcggct gcttctgtgg 60 cgg 63 28 63 DNA Artificial sequence Syntheticoligonucleotide 28 ccgccacaga agcagccgcc cttcttcttc ttcttcttctttcccccgca gtcacatgaa 60 cct 63 29 25 DNA Artificial sequence Syntheticoligonucleotide 29 tgccgagcca tcgacgtcag acgcg 25 30 30 DNA Artificialsequence Synthetic oligonucleotide 30 gcagatgtta acacacaagg cgttcttcca30 31 30 DNA Artificial sequence Synthetic oligonucleotide 31 ttgtgtgttaacatctgcgg tagctgcttg 30 32 29 DNA Artificial sequence Syntheticoligonucleotide 32 cagagagtta acagacaagc agctaccgc 29 33 30 DNAArtificial sequence Synthetic oligonucleotide 33 gtctgttaac tctctggaggttggtagata 30 34 30 DNA Artificial sequence Synthetic oligonucleotide 34acaaatgtta acattgaaaa ggtcatgatt 30 35 30 DNA Artificial sequenceSynthetic oligonucleotide 35 ttcaatgtta acatttgttt tctctgagcc 30 36 28DNA Artificial sequence Synthetic oligonucleotide 36 ggacgatatcgaaaagtttt ttcctcag 28 37 28 DNA Artificial sequence Syntheticoligonucleotide 37 acttttcgat atcgtccttg tggcttgc 28 38 30 DNAArtificial sequence Synthetic oligonucleotide 38 tctctggtta acccgggcccggccatggca 30 39 30 DNA Artificial sequence Synthetic oligonucleotide 39gcccgggtta accagagagt ctctgccatt 30 40 25 DNA Artificial sequenceSynthetic oligonucleotide 40 tgcgcagcca tcgacgtcag acgcg 25 41 31 DNAArtificial sequence Synthetic oligonucleotide 41 catgatgcat caaagttcaactgaaacgaa t 31 42 42 DNA Artificial sequence Synthetic oligonucleotide42 gatacttaag atctagtgga accaccacgc actcaaaggc tt 42 43 42 DNAArtificial sequence Synthetic oligonucleotide 43 ctagcttaag catgcatacaggtactggtc gatgagagga tt 42 44 25 DNA Artificial sequence Syntheticoligonucleotide 44 tgccgagcca tcgacgtcag acgcg 25 45 31 DNA Artificialsequence Synthetic oligonucleotide 45 catgatgcat caaagttcaa ctgaaacgaa t31 46 33 DNA Artificial sequence Synthetic oligonucleotide 46 cgagctcttcgatggctaca ggcagtggcg cac 33 47 36 DNA Artificial sequence Syntheticoligonucleotide 47 agcgctcttc ccatcgtatt agttcccaga ccagag 36 48 33 DNAArtificial sequence Synthetic oligonucleotide 48 cgagctcttc gacggctccgggaaaaaaga ggc 33 49 36 DNA Artificial sequence Syntheticoligonucleotide 49 agcgctcttc ccgtcttaac aggttcctca accagg 36 50 36 DNAArtificial sequence Synthetic oligonucleotide 50 cgagctcttc gatgcgtgcagcagctggag gagctg 36 51 36 DNA Artificial sequence Syntheticoligonucleotide 51 agcgctcttc gcatctcact gtcatcagac gagtcg 36 52 33 DNAArtificial sequence Synthetic oligonucleotide 52 cgagctcttc gacggctcctggaaagaaga gac 33 53 36 DNA Artificial sequence Syntheticoligonucleotide 53 agcgctcttc ccgtctcacc cgcttgctca accaga 36 54 36 DNAArtificial sequence Synthetic oligonucleotide 54 agttactctt ccatgacttcagttaattct gcagaa 36 55 36 DNA Artificial sequence Syntheticoligonucleotide 55 agttactctt ctttacaatg ggtgcacacg gctttt 36 56 36 DNAArtificial sequence Synthetic oligonucleotide 56 agttactctt cttaatcgtggacttaccgt ggatac 36 57 36 DNA Artificial sequence Syntheticoligonucleotide 57 agttactctt cccatcgtat tagttcccag accaga 36 58 29 DNAArtificial sequence Synthetic oligonucleotide 58 aagcgccgcg gccgctgcttatgtacgca 29 59 27 DNA Artificial sequence Synthetic oligonucleotide 59gacgcggaag cttcggtgga ctacgcg 27

That which is claimed is:
 1. A hybrid virus particle comprising: aparvovirus capsid; and an AAV genome packaged within said parvoviruscapsid, subject to the proviso that if said parvovirus capsid is an AAVcapsid, the serotypes of said AAV capsid and said AAV genome aredifferent.
 2. The hybrid virus particle of claim 1, wherein said AAVgenome comprises at least one AAV inverted terminal repeat.
 3. Thehybrid virus particle of claim 1, wherein said AAV genome is arecombinant AAV genome comprising at least one heterologous nucleic acidsequence.
 4. The hybrid virus particle of claim 1, wherein saidparvovirus capsid is an autonomous parvovirus capsid.
 5. The hybridvirus particle of claim 1, wherein said parvovirus capsid is a B19capsid.
 6. The hybrid virus particle of claim 3, wherein said AAV genomeis an AAV serotype-2 genome.
 7. The hybrid virus particle of claim 1,wherein said parvovirus capsid is an AAV capsid.
 8. The hybrid virusparticle of claim 7, wherein: said AAV genome is of a serotype selectedfrom the group consisting of AAV serotypes 1, 2, 3, 4, 5 and 6; and saidAAV capsid is of a serotype selected from the group consisting of AAVserotypes 1, 2, 3, 4, 5 and
 6. 9. The hybrid virus particle of claim 8selected from the group consisting of: (a) a hybrid virus particlecomprising an AAV serotype-3 capsid and an AAV serotype-2 genome, (b) ahybrid virus particle comprising an AAV serotype-4 capsid and an AAVserotype-2 genome, and (c) a hybrid virus particle comprising an AAVserotype-5 capsid and an AAV serotype-2 genome.
 10. The hybrid virusparticle of claim 1, wherein all of the AAV cap genes and all of the AAVrep genes are deleted from said AAV genome.
 11. The hybrid virusparticle of claim 2 comprising two AAV inverted terminal repeats thatflank said at least one heterologous nucleic acid sequence.
 12. Thehybrid virus particle of claim 3, wherein said at least one heterologousnucleic acid sequence encodes a protein or peptide.
 13. The hybrid virusparticle of claim 12, wherein said protein or peptide is a therapeuticprotein or peptide.
 14. The hybrid virus particle of claim 12, whereinsaid protein or peptide is an immunogenic protein or peptide.
 15. Thehybrid virus particle of claim 3, wherein said at least one heterologousnucleic acid sequence encodes an untranslated RNA.
 16. A pharmaceuticalformulation comprising the hybrid virus particle of claim 1 in apharmaceutically-acceptable carrier.
 17. An isolated nucleic acidencoding the hybrid virus capsid of claim 1, wherein said isolatednucleic acid comprises parvovirus cap genes and adeno-associated virus(AAV) rep genes, subject to the proviso that if said parvovirus capgenes are AAV cap genes, the serotypes of said AAV cap genes and saidAAV rep genes are different.
 18. The isolated nucleic acid of claim 17,wherein said parvovirus cap genes are operably associated with anauthentic parvovirus promoter.
 19. The isolated nucleic acid of claim17, wherein said parvovirus cap genes are B19 cap genes.
 20. Theisolated nucleic acid of claim 19, wherein said AAV rep genes are AAVserotype-2 rep genes.
 21. The isolated nucleic acid of claim 17, whereinsaid AAV rep genes encode at least one temperature-sensitive AAV Repprotein.
 22. The isolated nucleic acid of claim 17, wherein said capgenes are AAV cap genes.
 23. The isolated nucleic acid of claim 22,wherein said AAV cap genes are operably associated with an authentic AAVpromoter.
 24. The isolated nucleic acid of claim 23, wherein saidauthentic AAV promoter is an AAV p40 promoter.
 25. The isolated nucleicacid of claim 22, wherein: said AAV cap genes are of a serotype selectedfrom the group consisting of AAV serotypes 1, 2, 3, 4, 5 and 6; and saidAAV rep genes are of a serotype selected from the group consisting ofAAV serotypes 1, 2, 3, 4, 5 and
 6. 26. The isolated nucleic acid ofclaim 25 selected from the group consisting of: (a) a vector comprisingAAV serotype-3 cap genes and AAV serotype-2 rep genes, (b) a vectorcomprising AAV serotype-4 cap genes and AAV serotype-2 rep genes, and(c) a vector comprising AAV serotype-5 cap genes and AAV serotype-2 repgenes.
 27. A vector comprising the isolated nucleic acid of claim 17.28. The vector of claim 27, wherein said vector is selected from thegroup consisting of plasmids, naked DNA vectors, bacterial artificialchromosomes, yeast artificial chromosomes, and viral vectors.
 29. Thevector of claim 28, wherein said vector is a plasmid.
 30. A cellcomprising the vector of claim
 29. 31. The cell of claim 30, whereinsaid cell is selected from the group consisting of bacterial, protozoan,yeast, fungus, plant, and animal cells.
 32. A cell comprising a vectorcomprising: parvovirus cap genes, adeno-associated virus (AAV) repgenes, and an AAV genome, subject to the proviso that if said parvoviruscap genes are AAV cap genes, said AAV genome is of a different AAVserotype than said cap genes.
 33. The cell of claim 32, wherein saidcell is a mammalian cell.
 34. A cell comprising parvovirus cap genes andadeno-associated virus (AAV) rep genes stably integrated into the genomeof the packaging cell, subject to the proviso that if said parvoviruscap genes are AAV cap genes, the serotypes of said AAV cap genes andsaid AAV rep genes are different.
 35. The cell of claim 34 furthercomprising an AAV genome comprising, subject to the proviso that if saidparvovirus cap genes are AAV cap genes, the serotypes of said AAV capgenes and said AAV genome are different.
 36. A method of producing ahybrid virus particle, comprising: providing a cell withadeno-associated virus (AAV) rep genes, parvovirus cap genes, an AAVgenome, and helper functions for generating a productive AAV infection;subject to the proviso that if the parvovirus cap genes are AAV capgenes, the serotypes of the AAV cap genes and the AAV genome aredifferent, and allowing assembly of the hybrid virus particles.
 37. Themethod of claim 36, further comprising collecting the hybrid virusparticles.
 38. The method of claim 36, wherein the AAV genome comprisesat least one AAV inverted terminal repeat.
 39. The method of claim 36,wherein the AAV genome is a recombinant AAV genome comprising at leastone heterologous nucleic acid sequence.
 40. The method of claim 36,wherein the parvovirus cap genes and AAV rep genes are provided by oneor more transcomplementing packaging vectors.
 41. The method of claim36, wherein the parvovirus cap genes and AAV rep genes are provided by aplasmid.
 42. The method of claim 36, wherein the parvovirus cap genesand AAV rep genes are provided by an adenovirus vector.
 43. The methodof claim 36, wherein the AAV rep genes encode at least onetemperature-sensitive AAV Rep protein.
 44. The method of claim 36,wherein the parvovirus cap genes and AAV rep genes are stably integratedinto the genome of the cell.
 45. The method of claim 36, wherein theparvovirus cap genes are AAV cap genes.
 46. A hybrid virus particleproduced by the method of claim
 36. 47. A method of delivering a nucleicacid sequence to a cell, comprising: introducing into a cell a hybridvirus particle comprising a parvovirus capsid and an adeno-associatedvirus (AAV) genome packaged within the capsid, the AAV genome, subjectto the proviso that if the parvovirus capsid is an AAV capsid, theserotypes of the AAV capsid and the AAV genome are different.
 48. Themethod of claim 47, wherein the AAV genome comprises at least one AAVinverted terminal repeat.
 49. The method of claim 47, wherein the AAVgenome is a recombinant AAV genome comprising at least one heterologousnucleic acid sequence.
 50. The method of claim 49, wherein theheterologous nucleic acid sequence is expressed in the cell.
 51. Themethod of claim 47, wherein the parvovirus capsid is a B19 capsid. 52.The method of claim 49, wherein the at least one heterologous nucleicacid sequence encodes a protein or peptide.
 53. The method of claim 52,wherein the protein or peptide is a therapeutic protein or peptide. 54.The method of claim 50, wherein the protein or peptide is an immunogenicprotein or peptide.
 55. The method of claim 49, wherein the heterologousnucleic acid sequence encodes an untranslated RNA.
 56. The method ofclaim 47, wherein the cell is selected from the group consisting of aneural cell, lung cell, retinal cell, epithelial cell, muscle cell,pancreatic cell, hepatic cell, myocardial cell, bone cell, spleen cell,keratinocyte, fibroblast, endothelial cell, prostate cell, germ cell,progenitor cell, and a stem cell.
 57. The method of claim 47, whereinthe parvovirus capsid is an AAV capsid.
 58. The method of claim 57,wherein: the AAV genome is of a serotype selected from the groupconsisting of AAV serotypes 1, 2, 3, 4, 5 and 6; and the AAV capsid isof a serotype selected from the group consisting of AAV serotypes 1, 2,3, 4, 5 and
 6. 59. The method of claim 58, wherein the hybrid virusparticle is selected from the group consisting of: (a) a hybrid virusparticle comprising an AAV serotype-3 capsid and an AAV serotype-2genome, (b) a hybrid virus particle comprising an AAV serotype-4 capsidand an AAV serotype-2 genome, and (c) a hybrid virus particle comprisingan AAV serotype-5 capsid and an AAV serotype-2 genome.
 60. A method ofadministering a nucleic acid to a subject comprising administering thecell of claim 47 to a subject.
 61. A method of administering a nucleicacid sequence to a subject, comprising administering to a subject ahybrid virus particle comprising a parvovirus capsid and anadeno-associated virus (AAV) genome packaged within the capsid, subjectto the proviso that if the parvovirus capsid is from AAV, the serotypesof the AAV capsid and the AAV genome are different.
 62. The method ofclaim 61, wherein the AAV genome comprises at least one AAV invertedterminal repeat.
 63. The method of claim 61, wherein the AAV genome is arecombinant AAV genome comprising at least one heterologous nucleic acidsequence.
 64. The method of claim 61, wherein the subject is selectedfrom the group consisting of avian subjects and mammalian subjects. 65.The method of claim 62, wherein the subject is a human subject.
 66. Themethod of claim 62, wherein the subject is seropositive for the serotypeof the AAV genome.
 67. The method of claim 62, wherein the hybrid virusparticle is administered by a route selected from the group consistingof oral, rectal, transmucosal, transdermal, inhalation, intravenous,subcutaneous, intradermal, intracranial, intramuscular, andintraarticular administration.
 68. The method of claim 62, wherein thehybrid virus particle is administered to the liver of the subject. 69.The method of claim 68, wherein the hybrid virus particle isadministered to the liver by a route selected from the group consistingof intravenous administration, intraportal administration, intrabiliaryadministration, intra-arterial administration, and direct injection intothe liver parenchyma.
 70. The method of claim 63, wherein the at leastone heterologous nucleic acid sequence encodes a protein or peptide. 71.The method of claim 62, wherein the parvovirus capsid is an AAV capsid.72. A chimeric parvovirus capsid comprising at least one capsid regionfrom an adeno-associated virus (AAV) and at least one capsid region froma B19 virus.
 73. A chimeric parvovirus comprising the capsid of claim 72and an AAV genome.
 74. The chimeric parvovirus of claim 73, wherein saidparvovirus packages larger than wild-type AAV genomes.
 75. The chimericparvovirus of claim 73, wherein said parvovirus is about 33-38nanometers in diameter.
 76. The chimeric parvovirus of claim 73comprising an AAV capsid comprising a capsid B19 subunit.
 77. Thechimeric parvovirus of claim 76, wherein an AAV capsid subunit isreplaced by a B19 capsid subunit.
 78. The chimeric parvovirus of claim77, wherein the Vp3 subunit of the AAV capsid is replaced by the Vp2subunit of B19.
 79. A chimeric parvovirus capsid protein comprising atleast one capsid region from a different parvovirus.
 80. The chimericparvovirus capsid protein of claim 79, wherein an antigenic propertyrelated to the serotype of said parvovirus capsid protein is reduced ascompared with the wild-type parvovirus capsid protein.
 81. The chimericparvovirus capsid protein of claim 79, wherein said capsid protein is anadeno-associated virus (AAV) capsid protein.
 82. A chimeric virus capsidcomprising the chimeric parvovirus capsid protein of claim
 79. 83. Achimeric virus particle comprising: (a) a chimeric parvovirus capsid ofclaim 82; and (b) an AAV genome packaged within the chimeric parvoviruscapsid.
 84. The chimeric virus particle of claim 83, wherein said AAVgenome comprises at least one AAV inverted terminal repeat.
 85. Thechimeric virus particle of claim 83, wherein said AAV genome comprisesat least one heterologous nucleic acid sequence.
 86. The chimeric virusparticle of claim 83, wherein said capsid region from said differentparvovirus is inserted into said parvovirus capsid.
 87. The chimericvirus particle of claim 83, wherein said at least one capsid region fromsaid different parvovirus replaces a region within said parvoviruscapsid.
 88. The chimeric virus particle of claim 87, wherein said atleast one capsid region from said different parvovirus replaces ahomologous region within said parvovirus capsid.
 89. The chimeric virusparticle of claim 83, wherein said at least one capsid region from saiddifferent parvovirus is a loop region of the major capsid subunit. 90.The chimeric virus particle of claim 89, wherein said loop regionreplaces a loop region in the major subunit of said parvovirus capsid.91. The chimeric virus particle of claim 83, wherein said at least onecapsid region from said different parvovirus replaces a capsid subunitin said parvovirus capsid.
 92. The chimeric virus particle of claim 83,wherein said parvovirus capsid is an autonomous parvovirus capsid. 93.The chimeric virus particle of claim 83, wherein said parvovirus capsidis an adeno-associated virus (AAV) capsid.
 94. The chimeric virusparticle of claim 93, wherein an antigenic property related to theserotype of said AAV capsid is reduced as compared with the wild-typeAAV capsid.
 95. The chimeric virus particle of claim 93, wherein saidAAV capsid is a serotype-2 AAV capsid.
 96. The chimeric virus particleof claim 93, wherein said AAV genome is of the same serotype as said AAVcapsid.
 97. The chimeric virus particle of claim 93, wherein said AAVgenome is a serotype-2 AAV genome.
 98. The chimeric virus particle ofclaim 83, wherein said AAV genome is a serotype-2 AAV genome.
 99. Thechimeric virus particle of claim 83, wherein said different parvovirusis an AAV.
 100. The chimeric virus particle of claim 83, wherein saiddifferent parvovirus is an autonomous parvovirus.
 101. A pharmaceuticalformulation comprising said chimeric virus particle of claim 83 in apharmaceutically-acceptable carrier.
 102. An isolated nucleic acidencoding the chimeric virus capsid protein of claim
 79. 103. Theisolated nucleic acid of claim 102, wherein said at least one capsidregion is inserted into said chimeric capsid protein.
 104. The isolatednucleic acid of claim 102, wherein said at least one capsid regionreplaces sequences within said chimeric capsid protein.
 105. Theisolated nucleic acid of claim 102, wherein said chimeric parvoviruscapsid protein is a chimeric AAV capsid protein.
 106. The isolatednucleic acid of claim 105, wherein said isolated nucleic acid comprisesthe AAV cap genes and the AAV rep genes.
 107. A vector comprising theisolated nucleic acid of claim
 102. 108. A cell comprising the vector ofclaim
 107. 109. The cell of claim 108 further comprising anadeno-associated virus (AAV) genome.
 110. A cell comprising the isolatednucleic acid of claim 102 stably integrated into the genome of the cell.111. The cell of claim 110 further comprising an adeno-associated virus(AAV) genome.
 112. A method of producing a chimeric virus particle,comprising: providing a cell with parvovirus cap genes, rep genes froman adeno-associated virus (AAV), an AAV genome, and helper functions forgenerating a productive AAV infection; wherein the cap genes comprise atleast one nucleic acid sequence from the cap genes of a differentparvovirus; and allowing assembly of the chimeric virus particles. 113.The method of claim 112, further comprising collecting the chimericvirus particles.
 114. The method of claim 112, wherein the AAV genomecomprises at least one AAV inverted terminal repeat.
 115. The method ofclaim 112, wherein the AAV genome is a recombinant AAV genome comprisingat least one heterologous nucleic acid sequence.
 116. The method ofclaim 112, wherein the at least one nucleic acid sequence is insertedinto the parvovirus cap genes.
 117. The method of claim 112, wherein theat least one nucleic acid sequence replaces sequences within theparvovirus cap genes.
 118. The method of claim 112, wherein theparvovirus cap genes and AAV rep genes are provided by one or moretranscomplementing packaging vectors.
 119. The method of claim 112,wherein the parvovirus cap genes and AAV rep genes are provided by aplasmid.
 120. The method of claim 112, wherein the parvovirus cap genesand AAV rep genes are stably integrated into the genome of the cell.121. The method of claim 112, wherein the parvovirus is an AAV.
 122. Themethod of claim 121, wherein the AAV cap genes and AAV rep genes are ofthe same serotype.
 123. The method of claim 112, wherein the AAV repgenes are serotype-2 AAV rep genes.
 124. A chimeric virus particleproduced by the method of claim
 112. 125. A method of delivering anucleic acid sequence to a cell, comprising: introducing into a cell achimeric virus particle comprising a parvovirus capsid and anadeno-associated virus (AAV) genome packaged within the capsid, whereinthe parvovirus capsid comprises at least one capsid region from adifferent parvovirus.
 126. The method of claim 125, wherein the AAVgenome comprises and at least one AAV inverted terminal repeat.
 127. Themethod of claim 125, wherein the AAV genome is a recombinant AAV genomecomprising at least one heterologous nucleic acid sequence.
 128. Themethod of claim 127, wherein the at least one heterologous nucleic acidsequence encodes a protein or peptide.
 129. The method of claim 125,wherein the cell is selected from the group consisting of a neural cell,lung cell, retinal cell, epithelial cell, muscle cell, pancreatic cell,hepatic cell, myocardial cell, bone cell, spleen cell, keratinocyte,fibroblast, endothelial cell, prostate cell, germ cell, progenitor cell,and a stem cell.
 130. The method of claim 125, wherein the parvoviruscapsid is an AAV capsid.
 131. The method of claim 130, wherein the atleast one capsid region is from a B19 virus.
 132. The method of claim131, wherein the Vp3 subunit of the AAV capsid is replaced by the Vp2subunit of B19.
 133. The method of claim 125, wherein said AAV genome isa serotype-2 AAV genome.
 134. A method of administering a nucleic acidto a subject comprising administering the cell of claim 125 to asubject.
 135. A method of administering a nucleic acid sequence to asubject, comprising administering to a subject a chimeric virus particlecomprising a parvovirus capsid and an adeno-associated virus (AAV)genome packaged within the capsid, wherein the parvovirus capsidcomprises at least one capsid region from a different parvovirus. 136.The method of claim 135, wherein the AAV genome comprises at least oneAAV inverted terminal repeat.
 137. The method of claim 135, wherein theAAV genome comprises at least one heterologous nucleic acid sequence.138. The method of claim 135, wherein the subject is selected from thegroup consisting of avian subjects and mammalian subjects.
 139. Themethod of claim 136, wherein the subject is a human subject.
 140. Themethod of claim 135, wherein the subject is seropositive for theserotype of the AAV genome.
 141. The method of claim 135, wherein thechimeric virus particle is administered by a route selected from thegroup consisting of oral, rectal, transmucosal, transdermal, inhalation,intravenous, subcutaneous, intradermal, intracranial, intramuscular, andintraarticular administration.
 142. The method of claim 135, wherein thechimeric virus particle is administered to the liver of the subject.143. The method of claim 135, wherein the parvovirus capsid is an AAVcapsid.
 144. The method of claim 135, wherein the AAV genome is aserotype-2 AAV genome.
 145. The method of claim 135, wherein anantigenic property related to the serotype of said AAV capsid is reducedas compared with the wild-type AAV capsid.
 146. A targeted parvoviruscapsid protein comprising at least one exogenous targeting sequence,wherein said at least one exogenous targeting sequence confers analtered tropism to a virus particle comprising said targeted parvoviruscapsid protein.
 147. The targeted parvovirus capsid protein of claim146, wherein said parvovirus capsid protein is an autonomous parvoviruscapsid protein.
 148. The targeted parvovirus capsid protein of claim146, wherein said parvovirus capsid protein is an adeno-associated virus(AAV) capsid protein.
 149. The targeted parvovirus capsid protein ofclaim 146, wherein said at least one exogenous targeting sequence is acapsid sequence from an autonomous parvovirus.
 150. The targetedparvovirus capsid protein of claim 146, wherein said at least oneexogenous targeting sequence is a capsid sequence from an AAV.
 151. Thetargeted parvovirus capsid protein of claim 146, wherein said at leastone exogenous targeting sequence encodes a protein or peptide that bindsto a cell-surface receptor.
 152. The targeted parvovirus capsid proteinof claim 146, wherein said at least one exogenous targeting sequenceencodes a receptor ligand.
 153. The targeted parvovirus capsid proteinof claim 146, wherein a tropism of a virus particle comprising saidparvovirus capsid protein is reduced or eliminated.
 154. The targetedparvovirus capsid protein of claim 146, wherein a tropism of a virusparticle comprising said targeted parvovirus capsid protein is enhanced.155. The targeted parvovirus capsid protein of claim 146, wherein avirus particle comprising said targeted parvovirus capsid proteinacquires a new tropism.
 156. A virus capsid comprising the targetedparvovirus capsid protein of claim
 146. 157. The virus capsid of claim156, wherein said virus capsid is a parvovirus capsid.
 158. The viruscapsid of claim 157, wherein said parvovirus capsid is anadeno-associated virus capsid.
 159. A targeted virus particle comprisinga parvovirus capsid comprising: the virus capsid of claim 156, whereinsaid virus capsid is a parvovirus capsid; and an adeno-associated virus(AAV) genome.
 160. The targeted virus particle of claim 159, whereinsaid AAV genome comprises at least one AAV inverted terminal repeat.161. The targeted virus particle of claim 159, wherein said AAV genomeis a recombinant AAV genome comprising at least one heterologous nucleicacid sequence.
 162. The targeted virus particle of claim 159, whereinsaid AAV genome is a serotype-2 AAV genome.
 163. The targeted virusparticle of claim 159, wherein said parvovirus is an autonomousparvovirus.
 164. The targeted virus particle of claim 159, wherein saidparvovirus is an AAV.
 165. The targeted virus particle of claim 159,wherein said at least one exogenous targeting sequence is a capsidsequence from an autonomous parvovirus.
 166. The targeted virus particleof claim 159, wherein said at least one exogenous targeting sequence isa capsid sequence from an AAV.
 167. The targeted virus particle of claim159, wherein said at least one exogenous targeting sequence encodes aprotein or peptide that binds to a cell-surface receptor.
 168. Thetargeted virus particle of claim 159, wherein said at least oneexogenous targeting sequence encodes a receptor ligand.
 169. Thetargeted virus particle of claim 159, wherein said at least oneexogenous targeting sequence encodes an antibody or a fragment thereof.170. The targeted virus particle of claim 159, wherein said parvoviruscapsid and said AAV genome are of the same serotype.
 171. The targetedvirus particle of claim 159, wherein the AAV cap genes and AAV rep genesare deleted from said AAV genome.
 172. The targeted virus particle ofclaim 159, wherein a tropism of said parvovirus is reduced oreliminated.
 173. The targeted virus particle of claim 159, wherein atropism of said parvovirus is enhanced.
 174. The targeted virus particleof claim 159, wherein said parvovirus acquires a new tropism.
 175. Thetargeted virus particle of claim 159, wherein a tropism of saidparvovirus is reduced or eliminated and the parvovirus acquires a newtropism.
 176. The targeted virus particle of claim 159, wherein atropism of said parvovirus is reduced or eliminated and another tropismof said parvovirus is enhanced.
 177. The targeted virus particle ofclaim 159, wherein said at least one exogenous targeting sequenceencodes bradykinin or a fragment thereof.
 178. The targeted virusparticle of claim 164, wherein a nucleotide sequence encoding said atleast one exogenous targeting sequence is inserted or substituted intothe nucleotide sequence encoding the AAV capsid at a position selectedfrom the group consisting of nucleotide 2285, 2356, 2364, 2416, 2591,2634, 2690, 2747, 2944, 3317, 3391, 3561, 3595, 3753, 3761, 3766, 3789,3858, 3960, 3961, 3987, 4046, 4047 and 4160 of the AAV serotype 2genome, or the corresponding region of AAV of other serotypes, whereinthe inserted or substituted sequence begins at the nucleotide followingthe indicated position.
 179. The targeted virus particle of claim 164,wherein an exogenous targeting sequence is inserted or substituted intothe AAV capsid at a position selected from the group consisting of aminoacid 28, 51, 54, 71,130, 144, 163, 182, 247, 372, 396, 452, 464, 520,521, 517, 529, 552, 586, 595, 615, and 653 of the AAV serotype 2 Vp1capsid subunit and the corresponding position in the Vp2 and Vp3 capsidsubunits, wherein the inserted or substituted sequence begins at theamino acid following the indicated position.
 180. The targeted virusparticle of claim 179, wherein said virus particle consists of twoexogenous targeting sequences.
 181. A pharmaceutical formulationcomprising the targeted virus particle of claim 159 in apharmaceutically-acceptable carrier.
 182. An isolated nucleic acidencoding the targeted parvovirus capsid protein of claim
 146. 183. Theisolated nucleic acid of claim 182, wherein said parvovirus capsidprotein is an autonomous parvovirus capsid protein.
 184. The isolatednucleic acid of claim 182, wherein said parvovirus capsid protein is anadeno-associated virus (AAV) capsid protein.
 185. The isolated nucleicacid of claim 182, wherein said isolated nucleic acid encodes theparvovirus cap genes.
 186. The isolated nucleic acid of claim 185further comprising the AAV rep genes.
 187. A vector comprising theisolated nucleic acid of claim
 182. 188. The vector of claim 187,wherein said vector is selected from the group consisting of plasmids,naked DNA vectors, bacterial artificial chromosomes, yeast artificialchromosomes, and viral vectors.
 189. A cell comprising the vector ofclaim
 188. 190. The cell of claim 189 further comprising anadeno-associated virus (AAV) genome.
 191. A cell comprising the isolatednucleic acid of claim 182 stably integrated into the genome of saidcell.
 192. The cell of claim 191 further comprising an adeno-associatedvirus (AAV) genome.
 193. A method of producing a virus particle,comprising: providing a cell with parvovirus cap genes, rep genes froman adeno-associated virus (AAV), an AAV genome, and helper functions forgenerating a productive AAV infection; wherein said cap genes compriseat least one nucleic acid sequence encoding an exogenous targetingsequence; and allowing assembly of the virus particles containing saidat least one exogenous amino acid sequence, wherein said at least oneexogenous targeting sequence confers an altered tropism upon the virusparticles.
 194. The method of claim 193, further comprising collectingthe virus particles.
 195. The method of claim 193, wherein the AAVgenome comprises at least one AAV inverted terminal repeat.
 196. Themethod of claim 193, wherein the AAV genome is a recombinant AAV genomecomprising at least one heterologous nucleic acid sequence.
 197. Themethod of claim 193, wherein a virus stock is producing with a titer ofat least about 105 transducing units/ml.
 198. The method of claim 193,wherein a virus stock is produced with a titer of at least about 1transducing unit/cell.
 199. The method of claim 193, wherein the atleast one nucleic acid sequence is inserted into the parvovirus capgenes.
 200. The method of claim 193, wherein the at least one exogenousnucleic acid sequence replaces sequences within the parvovirus capgenes.
 201. The method of claim 193, wherein the parvovirus cap genesand AAV rep genes are provided by one or more transcomplementingpackaging vectors.
 202. The method of claim 201, wherein the parvoviruscap genes and AAV rep genes are provided by a plasmid.
 203. The methodof claim 193, wherein the parvovirus cap genes and AAV rep genes arestably integrated into the genome of the cell.
 204. The method of claim193, wherein the parvovirus cap genes are AAV cap genes.
 205. A virusparticle produced by the method of claim
 193. 206. A method ofdelivering a nucleic acid sequence to a cell, comprising: introducinginto a cell a targeted virus particle comprising a parvovirus capsid andan adeno-associated virus (AAV) genome packaged within the capsid, theparvovirus capsid comprising at least one exogenous targeting sequence,wherein the at least one exogenous targeting sequence confers an alteredtropism to the targeted virus particle.
 207. The method of claim 206,wherein the AAV genome comprises at least one AAV inverted terminalrepeat.
 208. The method of claim 206, wherein the AAV genome is arecombinant AAV genome comprising at least one heterologous nucleic acidsequence.
 209. The method of claim 206, wherein the cell is contactedwith a composition comprising at least about 106 transducing units ofthe targeted virus particle.
 210. The method of claim 206, wherein theat least one heterologous nucleic acid sequence encodes a protein orpeptide.
 211. The method of claim 206, wherein the cell is selected fromthe group consisting of a neural cell, lung cell, retinal cell,epithelial cell, muscle cell, pancreatic cell, hepatic cell, myocardialcell, bone cell, spleen cell, keratinocyte, fibroblast, endothelialcell, prostate cell, germ cell, prostate cell, progenitor cell, and astem cell.
 212. The method of claim 206, wherein the parvovirus capsidis an AAV capsid.
 213. The method of claim 206, wherein the tropism ofthe parvovirus for the cell is enhanced.
 214. The method of claim 206,wherein the parvovirus essentially does not infect or transduce the cellin the absence of the exogenous targeting sequence.
 215. The method ofclaim 206, wherein the at least one exogenous targeting sequence encodesbradykinin or a fragment thereof.
 216. The method of claim 206, whereinthe AAV genome is a serotype-2 AAV genome.
 217. A method ofadministering a nucleic acid to a subject comprising administering thecell of claim 206 to the subject.
 218. A method of administering anucleic acid sequence to a subject, comprising administering to asubject a targeted virus particle comprising a parvovirus capsid and anadeno-associated virus (AAV) genome packaged within the capsid, theparvovirus capsid comprising at least one exogenous targeting sequence,wherein the exogenous targeting sequence confers an altered tropism tothe targeted virus particle.
 219. The method of claim 218, wherein AAVgenome comprises at least one AAV inverted terminal repeat.
 220. Themethod of claim 218, wherein said AAV genome is a recombinant AAV genomecomprising at least one heterologous nucleic acid sequence.
 221. Themethod of claim 218, wherein the subject is administered a compositioncomprising at least about 106 transducing units of the targeted virusparticle.
 222. The method of claim 218, wherein the subject is selectedfrom the group consisting of avian subjects and mammalian subjects. 223.The method of claim 222, wherein the subject is a human subject. 224.The method of claim 218, wherein the virus particle is administered by aroute selected from the group consisting of oral, rectal, transmucosal,transdermal, inhalation, intravenous, subcutaneous, intradermal,intracranial, intramuscular, and intraarticular administration.
 225. Themethod of claim 218, wherein the virus particle is administered to theliver of the subject.
 226. The method of claim 218, wherein theparvovirus capsid is an AAV capsid.
 227. The method of claim 218,wherein the tropism of the parvovirus for a cell is enhanced.
 228. Themethod of claim 218, wherein the parvovirus capsid acquires a newtropism.
 229. The method of claim 218, wherein the AAV genome is aserotype-2 AAV genome.
 230. A virus particle comprising a parvoviruscapsid comprising at least one exogenous amino acid sequence, whereinsaid at least one exogenous amino acid sequence comprises a sequencethat facilitates purification of the virus particle.
 231. The virusparticle of claim 230, wherein said parvovirus is an adeno-associatedvirus (AAV).
 232. An adeno-associated virus (AAV) capsid comprising atleast one exogenous amino acid sequence is inserted or substituted intothe AAV capsid at a position selected from the group consisting of aminoacid 28, 51, 54, 71, 130, 144, 163, 182, 247, 372, 396, 452, 464, 520,521, 517, 529, 552, 586, 595, 615, and 653 of the AAV serotype 2 Vp1capsid subunit and the corresponding position in the Vp2 and Vp3 capsidsubunits, wherein the inserted or substituted sequence begins at theamino acid following the indicated position.
 233. The AAV capsid ofclaim 232, wherein the exogenous amino acid sequence encodes animmunogenic peptide or protein.
 234. The AAV capsid of claim 232covalently linked bound to, or encapsidating a compound selected fromthe group consisting of a DNA molecule, an RNA molecule, a protein, apeptide, a carbohydrate, a lipid, and a small organic molecule.
 235. AnAAV particle comprising the capsid of claim 232.